Motion modulation fluidic analyzer system

ABSTRACT

A fluid analyzer includes an optical source and detector defining a beam path of an optical beam, and a fluid flow cell on the beam path defining an interrogation region in a fluid channel in which the optical beam interacts with fluids. One or more flow-control devices conduct a particle in a fluid through the fluid channel. A motion system moves the interrogation region relative to the fluid channel in response to a motion signal, and a controller (1) generates the motion signal having a time-varying characteristic, (2) samples an output signal from the optical detector at respective intervals of the motion signal during which the interrogation region contains and does not contain the particle, and (3) determines from output signal samples a measurement value indicative of an optically measured characteristic of the particle.

BACKGROUND

Infrared spectroscopy is a valuable, well-known tool for chemicalcharacterization of gaseous, liquid and solid substances becausecompounds have distinct absorption “fingerprints” in the mid-infraredregion, with absorption bands corresponding to vibrational energies ofmolecular bonds. In theory, infrared spectroscopy should be a veryvaluable tool for analyzing liquid samples for applications including,but not limited to: medical liquid analysis (blood, urine, saliva, etc.)for diagnostics or substance detection; industrial or food/beverageprocess control; and pollutant detection.

A major barrier to broader application of infrared spectroscopy toliquid samples has been the high inherent absorption of many liquids inthe infrared. For example, water has strong infrared absorption, makinganalysis of aqueous solutions difficult. A number of tools have beendeveloped to circumvent this issue, for example: the use of attenuatedtotal reflection (ATR) prisms and other surface-grazing opticaltechniques; drying of samples before analysis; and the use ofliquid-liquid extraction processes to transfer solutes from one liquidto another, more infrared-transparent liquid. Each of these introducespotential complexities and inaccuracies into measurements of liquids.

One approach to address some of these limitations is to use new andimproved light sources in the infrared, including quantum cascade lasers(QCLs), that offer significantly higher power at specific wavelengths ofinterest than traditional globar (i.e. incandescent broadband thermalemitting) sources. This higher power potentially enables absorptionmeasurements in thicker liquid samples, while maintaining sufficientpower throughput to allow reasonable signal-to-noise for measurement ofchemical concentrations in the sample. Measurements can then beperformed with one or more wavelengths, with one or more “signal”wavelengths at absorption peaks of interest, and possibly wavelengthsdesigned to provide reference or baseline levels (off-peak). Multiplewavelengths may be achieved using multiple lasers, or through the use ofwavelength-tunable sources.

For detection of low concentrations of compounds in liquids, or subtlechanges in chemical makeup, the incremental infrared absorptioncorresponding to concentrations of interest may be extremely small.Therefore even with higher power transmission, there remains the problemof detecting small absorption signals against a high background.

One approach to measure low concentrations in spectroscopy is the use ofreference wavelengths. For example, sample transmission at thewavelength corresponding to an absorption peak of a substance ofinterest is measured, together with the transmission at two nearbywavelengths, one longer and one shorter. A “baseline” is then computedusing the reference wavelength transmissions, and the transmission atthe “peak” wavelength is divided by this baseline. This type of baselineadjustment can compensate for factors such as sample thickness, broadabsorption by other compounds, and detector responsivity changes. In thecase of broadband infrared sources, it also compensates—over a limitedwavelength range—for changes in source output. For example, suchreferencing will drastically reduce effects from changes in temperatureof a conventional blackbody thermal source. Indeed, this approach allowstraditional Fourier-Transform Infrared (FTIR) instruments equipped withglobar sources—or even using broadband radiation from synchrotronsources—to produce spectral data that may be locally baselined (inwavelength) to accurately determine chemical content.

Such baselining techniques, however, may be significantly less effectivewith infrared laser sources such as those that can deliver higher powerto penetrate thicker liquid samples. Laser sources are inherentlynarrowband, resonant devices, rather than broadband emitters. Theiroutput—power, wavelength, bandwidth, polarization and spatial beamproperties—can be highly sensitive to device and operating conditionsincluding current, temperature, aging, and feedback (from reflections).Moreover, any changes in these conditions may cause highly discontinuouschanges in output. Moreover, these changes will not be consistent fromone laser to another, or even from one wavelength to another in the caseof a broadband or tunable laser. As a result, changes betweenillumination at the “peak” (absorption, of a target compound) wavelengthand “reference” wavelengths may be very large compared to theincremental absorption from compounds of interest.

One method used for gaseous spectroscopy is the use of tunable lasersthat scan through an absorption peak in a short time. This is the coreconcept behind tunable diode laser absorption spectroscopy (TDLAS) thatis already used in commercial instruments. In gaseous samples,absorption peaks are typically very narrow (<<1 cm-1) and high. Thismeans a very narrow tuning range may be used (often <1 cm-1 inwavenumber terms) to cover reference and peak wavelengths. This tuningmay be performed quickly, and with minimum variation in laserconditions.

In liquid systems, on the other hand, absorption bands become farbroader, with lower peak absorptions. This requires tunable systems tocover a broader range (>10 cm-1 or even >100 cm-1, for example) which isdifficult to do consistently. For example, mode transitions within thelaser may occur inconsistently, leading to sharp changes in power,wavelength, and other beam characteristics at the wavelengths ofinterest. Similarly, multiple discrete sources operating at wavelengthsover the required range may individually vary in their emissioncharacteristics over time and operating conditions, leading to apparentchanges in “reference” and “peak” transmission and errors in reportedchemical concentrations.

Furthermore, although it is possible to integrate reference powerdetectors that monitor laser power prior to the sample, such referenceapproaches frequently require beam splitting optics which will introducenew optical artifacts such as fringing into the system. Thus the powersplit off by these optics may be different from the power delivered tothe sample as a result. In addition, such a reference channel will notaccount for optical effects within the sample and sample chamber—whichcan be particularly important in a coherent, laser-based system.

SUMMARY

A system is disclosed by which coherent light sources, including QCLs,may be used to measure liquid samples, and provides significantadvantages in terms of signal-to-noise ratio in measuring chemicalcomposition of these liquids. The system may be very stable in thepresence of laser and other optical train (path) and environmentalchanges. The system may also be used with non-coherent light sources.

The system includes an optical source and an optical detector defining abeam path of an optical beam, and a fluid flow cell disposed on the beampath defining an interrogation region in a fluid channel in the fluidflow cell in which the optical beam interacts with fluids. One or moreflow-control devices are configured to conduct a particle in a fluidthrough the fluid channel. A motion system moves the interrogationregion relative to the fluid channel in response to a motion signal, anda controller is configured and operative (1) to generate the motionsignal having a time-varying characteristic, (2) to sample an outputsignal from the optical detector at a first interval of the motionsignal during which the interrogation region contains substantially theparticle and at a second interval of the motion signal during which theinterrogation region contains a region substantially not containing theparticle, thereby generating corresponding output signal samples, and(3) to determine from the output signal samples a measurement valueindicative of an optically measured characteristic of the particle.

A microfluidic channel with laminar flow allows liquids to be presentedin nearly identical configurations to the light source, in closeproximity to one another, such that measurements of sample and referencefluids can be made within a short period of time during which the systemremains stable. In addition, the close proximity of the fluids to oneanother in a common flow ensures they may be presented in nearlyidentical conditions (pressure, temperature, flow rate, etc.).

In some embodiments, AC-coupled detectors may be used to measuredifferential absorption between reference and sample liquids at one ormore wavelengths as the scanning subsystem scans the beam between theliquid streams. The scan rate may be adjusted to optimize detector andsystem signal-to-noise ratio (SNR), for example by placing it above most1/f noise, but still within the acceptable response range for thedetector and its amplifying circuitry. Various well-known schemes forextracting and filtering a signal at a specific frequency may be used tooptimize SNR. Inherently, change-sensitive (“AC”) detectors such aspyroelectric detectors may be used, as may other thermal detectors suchas thermopiles, or photovoltaic detectors such as cooled or uncooledInGaAs or HgCdTe detectors. Pyroelectric detectors may provide anadvantage of high saturation flux (power per unit area) while remainingsensitive to small changes in infrared light as a result of differentialabsorption between reference and sample fluids.

Motion scanning may be achieved by scanning one or more beams opticallyover the microfluidic channel, or mechanically translating the samplerelative to the beam(s). Many subsystems for scanning beams over sampleshave been produced for microscopy, and similar subsystems may beutilized in the disclosed technique.

One or more infrared lasers may be used in the disclosed technique togenerate one or more wavelengths of interest, not limited to theinfrared. In some cases, a single fixed-wavelength laser could be usedto interrogate a specific absorption peak of a compound (i.e. analyte)that is not present in the reference liquid, but potentially present inthe sample liquid. As the beam scans between reference and samplefluids, the magnitude of the change detected on the detector allowscalculation of the concentration of the compound in the sample.

In other cases it may be helpful—because of interfering, non-targetcompounds, or because better concentration accuracy is desired—to usemultiple wavelengths, including at least one “peak” wavelength(measuring an absorption peak of interest) and one or more “off-peak”wavelengths. In such a configuration, these wavelengths may be deliveredsimultaneously from multiple lasers (which may be in a single-chiparray, or in discrete devices), or from one or more lasers that arewavelength-tunable. When multiple wavelengths are used simultaneously,these may be separated after transmission through the sample by means ofthin film filters, diffraction gratings, interferometers or similardevices. Relatively broadband laser sources, such as Fabry-Perot lasers,may be used, and component wavelengths split from one another opticallybefore detection. Alternatively, optical sources may be modulated inintensity in such a manner as to make their signals separable in thedetection system.

The disclosed technique may utilize any wavelengths and lasers thatresult in the desired measurement capability, including but not limitedto the UV, visible, near-infrared and mid-infrared regions where manycompounds have characteristic absorption peaks, but also in the THzrange where stronger optical sources such as QCLs are being developed.

The reference liquids used in the disclosed technique may be of severalforms. In the most basic configuration, the reference liquid consists ofa pure sample of the medium contained in the sample liquid—i.e.containing none of the target or analyte compound to be measured. Forexample, if the goal is to measure impurities (such as hydrocarbons) inwater, the reference liquid may be distilled water, or a known “clean”sample of water from the site being monitored.

In other cases, the reference liquid may contain the compound ofinterest at a desired concentration level; for example in an industrialprocess where a compound is added to a liquid medium, a reference liquidmixed to exact concentration in a laboratory may be used. Therefore anysignal detected as the beam in the system is scanned between sample andreference fluids indicates a deviation from the desired level. Thephase, or sign, of this signal will indicate whether there is too muchor too little of the compound, and magnitude will indicate the deviationlevel. As with many embodiments of the disclosed technique, multiplecompounds may be measured in this manner at multiple wavelengths. Forexample, an entire “panel” could be run in continuous, real-time fashionin a brewing process—against a “golden sample” of the product.Chemometric methods as known in the art may be applied.

In another example, a medical liquid such as blood plasma may beanalyzed with the disclosed technique against a standard reference thatcontains target levels of certain constituents, for example glucose orproteins. Any deviations may be measured with high contrast.

In other applications, the reference liquid may be a “before” sample,while the sample liquid is “after,” where chemical change is monitoredover time to measure degradation, for example. For instance, oilcondition in machinery or electrical equipment may be monitored in thismanner to track degradation and call for oil changes or otherpreventative maintenance. Again, the samples are presented in a laminarflow that allows nearly identical measurement conditions, and highcontrast and SNR resulting from the scanning measurement. The deviationsover time may be accumulated, providing both a change over a specifictime period as well as a cumulative deviation over multiple such timeperiods.

In other embodiments, the disclosed technique may be used in aconfiguration where a reference fluid is split into two streams, and onestream is exposed to gaseous, liquid, or solid samples that interactwith it (e.g. react with it, alter its chemical composition, orintroduce external compounds into it). The result of this interaction isnow the “sample” liquid, which is then measured as described above.Examples of such interactions include compounds dissolved from theexternal sample into the sample liquid, including liquid-liquidextractions, gas-to-liquid extraction, and solid-to-liquid extraction.For example, such a system may enable measurement of trace amounts of acompound on the surface of a solid, by first dissolving this compound ina known liquid, and then measuring the resulting sample liquid against apure sample of the liquid medium, with high contrast as describedherein. In other embodiments, through equivalency, the inverse may beperformed, that is a sample fluid is split into two streams, and onestream is exposed to gaseous, liquid, or solid media that react with it,alter its chemical composition, or extract external compounds from it.The result of this interaction is now the “reference” liquid, which isthen measured as described above.

In other embodiments, the sample liquid or stream may in fact consist ofthe analytes or intermingled fluids formed at the interface of twoliquids flowing in a laminar system, as a result of reactions betweenthose two liquids. In this case, the interface or boundary region, orregion of fluidic interaction, may be measured at various locations intothe flow chamber (e.g. by moving the interrogation region relative tothe fluid channel and measuring a boundary region signal from theoptical detector) in the direction of fluid flow in a microfluidicchannel, wherein the spatial position in the direction of fluid flow iscorrelated with the fluidic interaction time, and the reactionrates/concentrations or other characteristic of the boundary region arededuced from the rate of change of the infrared absorption signal fromthe sample stream. Thus the analyzer system and its controller may makeone or more measurements of the sample or reference, or sample andreference, and the interface region, and combine at least two of thosemeasurements to determine a characteristic of the interface region.

In other embodiments, the reference liquid may be pre-impregnated withcompounds other than those being measured, in order to facilitateaccurate measurement of liquid flow parameters. For example, it may bedesirable to measure the exact cross-section of the sample liquid vs.reference liquid in the laminar flow channel, in order to determinesample concentrations with maximum accuracy. For this purpose, thereference liquid, sample liquid, or both may include a marker that willbe missing from the other liquid or have a different concentration thanthe other liquid, allowing a difference to be detected by the analyzersystem. This marker may not necessarily have to function in theinfrared—it could be a color dye that is monitored optically in thevisible range and have absorption characteristics in the infrared thatdo not interfere with the desired measurement.

In other embodiments, the microfluidic cell or channel may be designedto interact with the sample fluid, or be the point of injection orexposure in order to create a difference between sample and reference.The sides of the optical interrogation channel may be coated with asubstance designed to create a differential reaction between sample andreference fluids, or the inlet channels to the cell may be similarlyprepared. The microfluidic cell environmental conditions may be altered(e.g. through its temperature) to create or enhance a difference betweensample and reference fluids. The microfluidic channel or channel sidesmay contain “posts”, “notches” or other flow modifying structures asknown in the art in order to induce desired or varying levels ofturbulent flow at the sample—reference fluidic interface region and thusmodify or enhance the signal in the interface region. The location ofthe fluidic boundary within the channel may be controlled or changed inorder to change or enhance the interaction between fluids, (e.g.different locations in the channel may provide different levels ofinteraction due to such structures or design elements within thechannel.). The analyzer may thus measure the fluidic boundary regionoptical characteristics at multiple points in the channel, each with adifferent level of fluidic mixing.

In certain applications, much of the reference liquid may be separatedand re-used at the end of the laminar flow section. Sufficient referenceliquid in the proximity to the sample liquid (enough to account fordiffusion or other phenomenon in the interaction region between fluids)may be stripped away and discarded, with the sample liquid, and theremainder of the reference liquid being recirculated, as for example maybe performed through microfluidic methods of directing differentportions of the channel fluid laminar streams into different outputchannels of the fluidic cell as is known in the art.

In other applications, the reference liquid and the sample liquid may beallowed to fully mix after measurement by continued diffusion or byother means known in the art. The mixed fluid may then be returned tothe source of the sample liquid to minimize sample loss instead of beingdisposed into a separate waste stream. This may be convenient in caseswhere the sample is highly valuable, or disposal may be undesirable dueto sample toxicity, or when it is desirable to operate in a closedsystem. In another embodiment, the level of fluid mixing may be knownand mixed fluid may be recirculated back to the fluidic cell for arepeat measurement at the mixed fluid concentration level, or forwardedto another fluidic analyzer cell for additional measurements. This mayenable the fluidic measurement system to dynamically dilute the sampleat known mixing ratios to obtain the optimum concentration level for themeasurement, or to dilute it to a desired level that is optimal forfurther measurements or requirements of a downstream process. Successivedilutions and re-measurement may be used to calibrate the fluidicanalyzer.

In another embodiment, the fluidic analyzer may be used as chemicalspecific detector for a liquid chromatograph. The reference fluid forisocratic solvent elution may be taken directly from the solventreservoir for the chromatographic pump. For liquid chromatographysystems that use gradient elution, the reference fluid is constantlychanging over the course of the chromatographic run and the referencefluid for the purposes of the invention should closely match the bulkcomposition of the solvent. This may be done by splitting the eluateoutput of the liquid chromatography column and using an analyte specificfilter to remove the analyte from the stream to generate the referencefluid. The reference fluid may then be measured against the unfilteredeluate in the fluidic analyzer. An example of such a filter may be amolecular sieve which would remove large molecules such a proteins.

In other embodiments, a single laminar stream of sample liquidsurrounded by reference liquid (either in 2 or 3 dimensions) isrequired. Such a laminar flow, and the methods and fluidic devices forproducing it, are well known from the fields of microfluidics andcytometry.

In still other embodiments, it may be advantageous to produce amultiplicity of laminar sample and reference streams with a multiplicityof fluid boundary regions, alternating across the flow channel. Such aconfiguration may allow higher SNR in the signal resulting from motionscanning, such as by changing the optical signal modulation frequency atthe detector to be higher than the frequency of a controller signal thatdrives a repetitive motion of the interrogation region relative to thechannel or fluid boundary regions.

For high transmission in the infrared, it may be desirable to userelatively thin flow channels, for example <1 mm, or in many cases <100microns (um), <50 um, <25 um or even <10 um—depending on thetransmission of the fluid, and the fluid dynamic parameters required tomaintain a laminar flow.

The scanning beam and surface angles of the fluidic chamber may bearranged so as to minimize surface reflections which may interfere withmeasurements by variable constructive or destructive interference andeven potentially feedback to the laser. As most infrared laser sourcesare inherently polarized, the surfaces may be oriented such that Ppolarized light experiences no reflection as it passes through themeasurement chamber.

The disclosed technique may utilize either transmissive or transflective(where light passes through the liquid, reflects, re-passes through theliquid and then back to a detector) configuration.

The disclosed technique may incorporate surface-grazing/evanescentcoupling absorption spectroscopy techniques such as the use of photoniccrystals that are in contact with the sample and reference fluid flowsor, more commonly, ATR prisms, where the measurement face forms one sideof the fluid flow channel. In such architecture, scanning is stillachieved by moving the beam (which enters the ATR, and reflects at leastonce off the surface in contact with the fluid) perpendicularly relativeto the laminar fluid flow over the measurement face of the ATR crystal.

The disclosed technique may be used throughout the visible, infrared andterahertz range where laser sources are available. Specifically, it maybe used in the near-infrared (0.75-1.4 um), short-wave infrared (1.4-3um), mid-wavelength infrared (3-8 um), long-wavelength infrared (8-15um), and far-infrared (20-1000 um). These are regimes where compoundshave characteristic vibrational absorption lines and laser as well asdetector components have been developed, capable of being used asdescribed above.

Quantum cascade lasers (QCLs) may offer specific advantages for use inthe disclosed technique. They may be fabricated to emit at wavelengthsthroughout the mid-infrared as well at the terahertz ranges where thedisclosed technique may be used to measure liquid properties. They areavailable in multiple formats, including discrete narrowbandsingle-wavelength devices, broadband (Fabry-Perot) emitters which mayoptionally be combined with wavelength-selective or dispersive elementsto select one or more specific wavelength bands, wavelength-tunablesubsystems, and QCL arrays which may emit a number of wavelengths from asingle-chip device. All of these forms of QCL may be used in the contextof the disclosed technique.

Applications

Applications of the disclosed technique include, but are not limited to:

-   -   measurement of medical fluids including blood plasma, urine, or        saliva against standard reference fluids for diagnostic        purposes, or to monitor for controlled substances; this may        include the measurement of blood glucose level;    -   measurement of water samples against reference water samples to        test for or determine concentrations of pollutants;    -   measurement of biological samples against reference media to        measure levels of DNA, RNA, proteins, sugars, lipids, cellular        nutrients and metabolites; this includes measurement of liquids        which have surrounded cells or tissue (such as cancer cells,        stem cells, embryos) to measure uptake of nutrients and/or        production of metabolites; measurement of DNA levels in        polymerase chain reaction (PCR) tests;    -   measurement of large molecule biologics such as proteins,        carbohydrates and lipids to determine their higher order        molecular structure;    -   measurement of liquid samples from food, drink, or        pharmacological production processes against standard reference        liquids to provide feedback for production parameters, measure        completion, or measure contamination;    -   measurement of liquids used in electrical or mechanical        machinery against standard reference liquids to measure wear and        schedule preventative maintenance or replacement;    -   measurement of airborne chemicals through trapping in a liquid        stream, and comparison to a pure reference liquid;    -   measurement of chemical composition in solids through exposure        to a liquid, and comparison of that liquid to a pure reference        liquid;    -   measurement of the eluate fluids at the output of a liquid        chromatography instrument    -   measurement of liquids such as milk against a standard reference        to determine nutritional and fat content, and other parameters;        measurement of potable liquids such as olive oil against a known        reference to determine authenticity and purity; measurement of        potable liquids against reference liquids to measure potentially        harmful impurities.

More generally, the disclosed technique may be extended to allowmeasurement of liquid-based samples either in flow or non-flowenvironments. The essential elements remain the same: and infrared lasersource such as a QCL (which may operate at one or more wavelengths), mayinclude concentration gradients (the target of this measurement system)which result in a variation in the extinction of infrared light as itinteracts with the sample (where the aforementioned scanning convertsthis spatial variation into a temporal variation in a specific frequencyrange), a mechanism to guide the resulting infrared light (afterscan-modulated interaction with the sample) to a detector subsystemwhich includes an AC-sensitive detector designed to measure changes ininfrared light intensity corresponding to the scanning frequency range;the output of this detector subsystem being used to calculateconcentrations of target substances in the liquid. This scanning may beperformed in 1- or 2-dimensions on a liquid-based sample as describedbelow.

For purposes of clarification, the term sample may refer to a substanceto be measured in the analyzer (e.g. a fluid, a fluid with analyte(s),etc.) or when used in the context of a differential or multiple samplemeasurement wherein the measurements are combined to determine a fluidcharacteristic, sample may refer to a fluid other than a reference fluid(e.g. sample and reference fluids). The sample fluid in this instancemay also be referred to as the analyte fluid or analyte sample (e.g.analyte and reference fluids).

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages will beapparent from the following description of particular embodiments of theinvention, as illustrated in the accompanying drawings in which likereference characters refer to the same parts throughout the differentviews.

FIGS. 1A-1E are a set of plots of optical spectra;

FIG. 2 is a schematic diagram of a flow channel;

FIGS. 3A-3D are a set of plots of position, power, voltage andcalculated concentration as functions of time;

FIG. 4 is a block diagram of a fluid analyzer;

FIGS. 5-7 are block diagram of fluid analyzers;

FIG. 8 is a schematic diagram of a sample holder;

FIGS. 9A-9B show an example of a liquid chamber/channel-integratedattenuated total reflection (ATR) prism;

FIG. 10A illustrates a use in which a scanned liquid sample includesdispersed solids or liquids;

FIG. 10B is a block diagram of a fluid analyzer for a use like that ofFIG. 10(a);

FIGS. 11A-11D are a set of plots of various parameters as a function offrequency;

FIG. 12 is a schematic diagram of a fluid analyzer;

FIGS. 13 and 14 are schematic diagrams of a fluid flow cell;

FIGS. 15 and 16 are schematic diagrams of a fluid analyzer;

FIGS. 17-20 are schematic diagrams of fluid flow cells.

DETAILED DESCRIPTION

FIGS. 1A-1E are several graphs or plots showing certain illustrativeoptical spectra.

FIG. 1A shows absorption of a target compound, in its pure form, as afunction of frequency v. In this simplified example, a single absorptionpeak is shown.

FIG. 1B shows the absorption of a medium in which the compound oranalyte is dissolved; in this case, a uniform high absorption is shown(which is the case, for example, for water over certain infraredranges). Note the liquid medium may have a very complex absorptionprofile with multiple absorption peaks, and may consist of manyintermingled chemical components. The disclosed technique is very wellsuited to handle such scenarios where the medium has complex absorptionpatterns, as it inherently removes common components between a referenceand sample fluid, and therefore the features of the medium in which thetarget compound is carried (e.g. the example where the target is insolution).

FIG. 1C shows the transmission through the liquid sample, including boththe medium and target compound. Note the overall transmission may bevery low (as is the case with aqueous solutions in the mid-infrared),and the incremental absorption due to the compound of interest may beextremely small. Moreover, with a broadband infrared source such as aglobar or even synchrotron, the power density per unit frequency is verylow, so the total power delivered to the frequency range where thecompound absorbs is very low. This makes accurate measurement of samplesin liquid very challenging using conventional sources.

FIG. 1D shows an ideal situation in which three narrowband infraredlaser sources are used to measure reference and signal absorptionfrequencies, compute peak absorption, and thereby determineconcentration of the compound.

FIG. 1E shows a more realistic operation of such systems—the laser powermay vary significantly over frequency, as may its bandwidths/bandshapes, spatial modes, etc. These characteristics may also varysignificantly with time, temperature, vibration/shock, and otherenvironmental parameters. This means the variation in lasercharacteristics overwhelms differential absorption from the compound ofinterest in many cases, even when great lengths are taken to stabilizeor calibrate the system.

FIG. 2 shows a system that overcomes such issues by presenting theliquid sample in a flow configuration that allows referencing against astandard. In this system, a sample fluid 201 flows into a chambertogether with one or more reference fluids 202 with laminar flow 203. Inthe optical measurement region 204 a beam is scanned across thereference fluid 201 as well as sample fluid 202, with at least oneregion 205 where it substantially measures absorption of the samplefluid 201, and one region 206 where it substantially measures absorptionin the reference fluid 202.

In the arrangement of FIG. 2, a laminar flow is established whichcombines the sample fluid with a reference fluid, and these flow side byside through the optical measurement interrogation zone or region withina fluidic channel. In the measurement zone, an infrared beam isrelatively translated (scanned) back and forth over the reference andsample liquids. A laminar flow system, which may be a microfluidicsystem in many applications, ensures that there is not strong mixingbetween the sample and reference liquids; the parameters for such a flow(dimensions, flow rate) are well established in the art. The measurementzone may be set in a region where there is a stable flow and wheresignificant diffusion of the compound(s) of interest between the sampleand reference of measurement significance has not occurred (however, insome cases, this may be desirable, as noted above). The motion scanningrange should be large enough to optically sample the sample andreference fluids adequately, but typically limited in range in order tomaintain substantially identical optical path conditions in the system.In some embodiments the microfluidic channel itself may be motiontranslated across the beam, while in other embodiments the beam will bescanned over the channel. In other embodiments, a fluidic chamber may bepre-charged with a laminar flow, the flow terminated, and then thechamber measured optically before significant diffusion occurs. Thechamber itself may be part of a disposable unit, built using low-costmicrofluidic manufacturing techniques. This unit may include thereference liquid on-board, as well as in some cases any liquid requiredto prepare the sample fluid. Note that while the flow cell shown in FIG.2 has two reference flows on either side of a sample flow (which isoften helpful for “centering” the flow), but other configurations arepossible. One embodiment would merge one sample liquid flow with asingle reference liquid flow (i.e. 2 input flows), and scanning wouldoccur proximate to the interface of these streams. More complex flowsmay include multiple reference and sample flows interleaved.

FIGS. 3A-3D are several graphs or plots representing the operation ofthe system as it is used to determine concentration of a target compoundin the sample fluid, in this embodiment using a single infrared lasersource and single detector.

FIG. 3A shows an example of a scanning pattern as may be generated froma system controller scanning modulation waveform (triangular in thiscase, though many other known optical scanning patterns may be used,including 2-dimensional scan patterns) where the infrared beam isscanned from reference fluid, through sample fluid, and back toreference fluid. Note that the optical beam does not necessarily need topass across the entire width of the sample stream; it could simplyoscillate on one edge of the flow between sample and reference fluids. Afeedback loop may be used to continuously center the scan optimally onthe edge or center of the sample flow—this feedback may use theabsorption of the compound of interest, or other unrelated absorptionpeaks that are always present (including reference compounds added tothe reference or sample liquid, as described above) as a measurementparameter in the feedback loop. The analyzer may include a transducerfor detection of the position of the fluid interface (i.e. boundaryregion) or interfaces and generating a signal for determining the timingof the sampling of the detector output signals, such as may beperformed, by way of example, with a conductivity sensor if the fluidshave different conductivities, or optically if the fluids have different“colors” at visible wavelengths.

FIG. 3B shows the transmitted optical power as the beam is scanned overthe channel, at three different concentration levels. The incrementalabsorption as the beam passes over the sample may be extremely small.Note in some embodiments, the disclosed technique may in fact be used tomeasure the absence or reduction of the absorption peak in the samplefluid.

FIG. 3C shows the output of an example detector circuit in response tothese optical transmission changes. The detector and/or circuit areconfigured in this case to use an AC detection mode, where only changesin optical power are registered (as the derivative of that power withtime). Such a configuration may provide significant advantages where theincremental absorption is very small—it effectively removes the highbaseline, and any common absorption features. Note that in some caseswhere the absorption of the target compound is high, a conventional DCdetection scheme may be used. Even when an AC detection scheme is used,it may be useful to measure DC power, either with the same detector(through a split AD/DC circuit) or with a separate detector, so as tonormalize the AC signal by the DC optical power (which will take intoaccount laser power and overall liquid and system transmission, amongother long-term changes). AC detectors such as pyroelectric detectors,which are low-cost and are stable over temperature, may be employed inthe disclosed technique, as may the whole class of well-known detectorsand circuits that have been developed for FTIR instruments (whichmeasure AC signals resulting from a scanning interferometer).

FIG. 3D shows the concentration of the target compound calculated in thecurrent system. This concentration could be calculated from a singlescan, or from the aggregate of many scans, depending on the accuracy andreal-time characteristics required for the application.

FIG. 4 presents a generalized version of the disclosed technique. Amid-infrared laser source (SRC) 401 produces mid-infrared light 402 thatis motion scanned relative to a sample chamber 405 by motion controldevice, realized by a motion scanning system (SCAN) 403 in theillustrated embodiment. Motion scanning is also referred to as “motionmodulation” herein. The scanning system 403 may be a system thattranslates the sample chamber in relation to a stationary optical beam.Here the scanning system 403 is shown to scan the optical beam over arange of positions 404 that pass through the chamber (e.g. microfluidiccell) windows 406 and the contained liquid sample 407 in a chamberregion (e.g. microfluidic channel). As the beam is scanned throughdifferent portions of the liquid sample (the region of opticalinteraction of the optical beam and fluid being referred to as theinterrogation region), which may contain one or more parallel streams ofsample and reference fluids or combinations of both, the amount ofmid-IR light transmitted at specific wavelengths may vary by transmittedbeam position 408. A de-scanning mechanism (DE-SCAN) 409 serves todeliver all of this light substantially to a detector subsystem (DET)411. The de-scanning mechanism 409 may be one and the same as thescanning mechanism 403, in the case where the sample chamber istranslated to achieve the scanning, or in some cases a lens withappropriate characteristics may be used to focus substantially all thescanned light onto the detector element. The de-scanned light 410reaching the detector subsystem 411 therefore is modulated by scanningit through the liquid sample 407, with all other conditions heldsubstantially identical through the course of the scan. The detectorsubsystem 411 is an AC-coupled detector system that either uses adetector such as a pyroelectric detector which is responsive only tochanges in optical power, and/or may employ a circuit to remove any DCcomponent of the mid-infrared signal 410 reaching the detectorsubsystem. Therefore gain can be applied in order to amplify effectsfrom small changes in transmission due to scanned concentrationgradients, without saturating the output of the detector subsystem. Theoutput 412 of the detector subsystem is processed by a computing unit orcontroller (COMP UNIT) 413 that calculates a measurement value to, insome embodiments, determine an optically measured characteristic (e.g.analyte concentration) of the fluid 414. The optically measuredcharacteristics may be calculated as a function of position in themicrofluidic channel when multiple measurement points are taken duringthe scanning. The controller may also generate a motion control signalfor the purpose of driving the motion control device to create themotion modulation or movement.

The term measurement value as used herein generally refers to a valuedetermined by the modulation or change of the optical power incident onthe detector as a result of at least two of the sample fluid, referencefluid, fluid interface boundary region, or particle being within theinterrogation region. The measurement value may then be combined withoptical pathlength, optical power, or some other parameter of theanalyzer to determine an optically measured characteristic of thefluids, interface region or substance within the fluids (e.g. an analyteconcentration) In some embodiments of the invention, the measurementvalue may be the optically measured characteristic (e.g. in a biologicalprocess feedback system designed to control the signal modulation on thedetector to a desired level, which may vary over time or fluidenvironmental conditions, the modulation signal level indicative of atarget analyte concentration or other property through prior calibrationof the analyzer).

In one embodiment, core elements of a disclosed system are: the use ofmid-infrared lasers such as QCLs to produce light at wavelengthscorresponding to compounds of interest in the liquid-based sample; amethod of scanning this light relative to the sample in order tomodulate transmission according to local concentrations of thesecompounds; a method of delivering the transmitted light to an AC-coupleddetector system which amplifies these transmission differentials thatresult from scanning; and a system controller to compute absorption andpotentially relative concentrations within the sample.

Examples of detectors types include mercury-cadmium-telluride (MCT)photoconductive or photovoltaic detectors run in AC-coupledamplification circuits, or pyroelectric detectors which are inherentlyAC-coupled in nature. For many applications, pyroelectric detectors maybe well suited because of their AC-coupled nature, very high saturationpower, temperature insensitivity, and low cost. Importantly,pyroelectric detectors remain linear over a wide range of powers(whereas MCT detectors saturate). In particular in a case wheremid-infrared lasers are used, there is often plenty of power, and thedisclosed technique allows concentration measurements through thedetection of small changes in this power (rather than absolute DC powermeasurement).

The detector subsystem 411, in addition to the AC-coupled primarydetector(s), may additionally comprise a DC level detector that monitorsthe overall transmitted mid-IR light, and is used to normalize the ACsignal. Such DC-level detection allows adjustment for overall laserpower, system transmission, liquid sample thickness, etc.

While many embodiments may use a transmission-type design where thescanned beam (where “scanned” is understood to mean either the beamscanning over the fluid stream, or the fluid stream being scanned (e.g.moved) relative to the optical beam) is transmitted through the samplechamber and the sample. However, the disclosed technique extends todesigns employing “transflection” (where the beam passes through thesample, is reflected, and passes through the sample once more on itspath to exit), as well as surface-sampling techniques such as attenuatedtotal reflection (ATR) prism-based designs where the beam reflects off asurface in contact with the liquid sample and evanescently couples intoit, evanescent waveguide designs, and designs where resonant surfacecoatings (such as photonic crystal or metamaterial designs) in contactwith the sample amplify interaction between the mid-infrared light andthe sample.

The beam scanning frequency and pattern may vary by configuration andapplication. In one embodiment, the scanning may allow the signalcorresponding to the absorption, and therefore the concentrationgradient, to be shifted to a frequency well above low-frequency noisesources (e.g. 1/f noise) and variations in the system (e.g. temperaturefluctuations in the mechanics or laser, etc.) and thereby avoid many ofthe disadvantages of static (DC) transmission measurements systems. Forexample, the scanning frequency may be at least nominally 1 Hz, 10 Hz,100 Hz, 1000 Hz, 10000 Hz or higher as the motion and detectorsubsystems allow. The scanning frequency may also fall into a rangewhere the detector employed has sufficient response. For example,pyroelectric detectors are thermal detectors, and therefore have aroll-off in signal with frequency that may be pronounced over 100 Hz.The detector circuit may also be designed and optionally optimized forthe scanning frequency. Well-known “lock-in amplification” techniquesmay be applied to isolate the signal resulting from the scanning; thephase of the detected signal relative to the scanning may be used tofurther refine the signal. For example, in cases where a known interfacebetween two fluids (say, side-by-side laminar flows of a sample andreference fluid) is scanned, the change in transmitted intensity at thatinterface may be isolated from other scanning-related optical artifacts.Alternatively, a baseline may be established by running the scan over asection of sample known to have no concentration gradients. Variousother digital filtering techniques that are well known in the art may beapplied after the amplified detector signal is captured and digitized.

FIG. 5 shows another embodiment of the disclosed technique. Amid-infrared laser source (SRC) 501 (which may produce one or morewavelengths in the mid-infrared) is focused by a lens 502 through aspatial filter 503 which is designed to “clean up” or optimize themid-infrared beam, with the transmitted light well-suited for focusinginto a well-defined spot (despite any variation in the output of thelaser, such as different spatial modes); the filtered light isre-collimated by lens 504 and then scanned over a range of angles byscanner 505. The scanner may scan in 1 or 2 axes. The scanned light isfocused by lens 506 onto sample holder 507 (e.g. microfluidic cell withfluid channel). The scanned beam 507 c (showing two beam positionswithin the scan) passes through the sample chamber windows 507 a and thecontained liquid-based sample 507 b (which in this embodiment, shows tworegions with differing concentration of a target compound). The sampleholder may optionally be mounted on a translation stage 508 with one ormore translation axes in order to position the sample relative to thescanning beam. For example, a “Z” translation (substantially parallel tothe axis of the beam) may be used to optimally focus the beam on thesample within the sample holder for best measurement resolution, andthereby get maximum contrast during the scan; “X” and or “Y” translationmay be used to position the sample such that the scanning beam traversesspecific features having concentration gradients of interest (forexample, the boundary between two liquid flows). A capturing lens 509re-collimates the transmitted mid-IR light and a de-scanning mirror 510redirects the mid-IR light such that the light remains incident on thedetector 513 with minimal intensity modulation when there is noconcentration gradient in the sample. A lens 511 focuses the light,optionally through a spatial filter 512, onto the AC-coupled detectorsystem (DET) 513. The detector signal(s) are relayed to a computingcontroller unit (COMP UNIT) 514 that computes absorption gradients, andpotentially concentrations of analytes, in the sample. The computingcontroller unit 514 may also control laser operation (power andwavelength, for example), scanning and de-scanning modules, andtranslation stage(s), and generate scan modulation waveforms.

The disclosed technique may be used to measure liquid-based samples ofvarious types, including liquid flows with concentration gradients, anddispersions of droplets or solid particles in liquids. Each sample willideally have concentrations gradients over the scale scanned by thedisclosed technique, so as to induce a change in the amount of lighttransmitted, and therefore an AC signal on the detector. The change insignal may in fact result from the displacement of the medium (forexample, water) by a solute or dispersed material, or scattering as aresult of the difference of refractive index between a droplet or solidparticle and the surrounding medium.

In some embodiments, the disclosed technique may measure or calculatescattering resulting from particles or droplets dispersed in the liquidsample—again by scanning between regions with more and fewer of suchparticles or droplets, or between regions where such particles ordroplets change in nature. In such embodiments, scattering may increaseas a function of droplet or particle diameter or refractive index, whichis a function of composition and wavelength. Through the use ofappropriate spatial filters before and after the sample, it is possibleto isolate or remove scattered light, and thereby calculate scatteringfrom particles or droplets in the liquid in order to deduce averagediameter (assuming some chemical composition). With multiple wavelengthsaround infrared absorption peaks for droplet/particle constituents, itis additionally possible to estimate both chemical composition as wellas droplet size as it results from resonant Mie scattering (that is,rapid change in scattering as a result of rapid change in refractiveindex around a resonant absorption peak for a particular compound).

For example, in measurements of hydrocarbons in water, some hydrocarbonsmay not be dissolved in the water but form droplets dispersed in thewater. The disclosed technique may be used to measure a sample of waterwith potential hydrocarbon analytes in a laminar flow side-by-side witha pure water reference, by scanning the beam (or equivalently, thesample chamber) back and forth across the interface between theseparallel flows. Measurements may be made at several wavelengths,including a peak absorption wavelength for hydrocarbons, but also anon-peak wavelength. Non-peak wavelength signal may indicate scatteringand water displacement; the differential between peak and non-peak mayindicate hydrocarbon concentration. Additionally, if wavelengths oneither edge of the absorption peak are measured, the differential inscattering loss (as a result of resonant Mie scattering) may be used tocalculate dispersed hydrocarbon characteristics. Therefore the disclosedtechnique may be used to measure both dissolved and dispersedhydrocarbons in a water sample, and distinguish between these.

FIG. 6 shows another embodiment of the disclosed technique; this exampleshows a system where “de-scanning” onto a single detector element isaccomplished with the use of a short focal length lens 609. A completedescription is as follows: a mid-infrared laser source (SRC) 601 such asa QCL (which may be a single-wavelength device, emit multiplewavelengths, or have a tunable wavelength) is collimated through lens602 (all lenses described herein may be refractive or reflective-typelenses), and then scanned using scanner 603 over a range of angles,before being focused on the liquid sample chamber 606 by lens 604. Thesampling spot therefore is scanned over a section of the liquid sampleas indicated by 605; upon transmission through the liquid sample it maybe differentially attenuated depending on chemical concentrations withinthe sample and the interrogating wavelength(s); the beam scanningconverts such gradients into a periodic power fluctuation in thetransmitted light. A collimating lens 607 re-collimates the light, andin this example a fixed folding mirror 608 redirects the collimated beamto a short focal length lens 609. The function of the short focal lengthlens is to focus the transmitted infrared light onto the detector (DET)610. Generally a small detector area is desired, as noise grows with thesquare root of area. In this example, a short focal length is used atthe detector compared to the focal length of focusing lenses 604,607. Asa result, the motion of the beam spot on the detector will be a fractionof the motion of the spot on the sample, allowing a reasonable scandistance on the sample while maintaining focus on the surface of a smalldetector. The signal from the detector subsystem may be used by acomputer unit (CU) 611 to calculate absorption and possiblyconcentrations and other fluid characteristics, which go to output 612.

In some embodiments it may be desirable to use detectors with asymmetricdimensions (for example, an elongated rectangle), and to orient thisdetector with its long axis along the scan direction, to facilitatecomplete (or at least consistent) beam capture throughout the scancycle. In some cases detector arrays may be used in the disclosedtechnique; however, the scanning would not typically result in beamspot(s) moving from detector element to detector element (which wouldcause very large signal swings not related to concentration gradients inthe sample). In other embodiments, the optical beam with a generallyelliptical spatial form may have its long axis parallel to the directionof fluid flow and its short axis parallel to the direction of motionscanning of the optical beam relative to the sample chamber.

In other embodiments, multiple beam spots may be used and scannedsimultaneously across the sample. These may be multiple spots ofidentical wavelength, split in order to take advantage of increasedperformance from the use of an array of detectors (where the light fromeach beam remains focused on its corresponding detector elementthroughout the modulating scanning described herein). Alternatively, ifan infrared laser array such as the distributed feedback (DFB) QCLdescribed by Capasso et al is used, each spot may correspond to adifferent wavelength of interest, and may be relayed to itscorresponding detector after interacting with the sample. In anotherembodiment, the more than one spot passing through the fluid stream maybe directed to a single detector.

In one embodiment, a QCL DFB array with wavelengths corresponding to oneor more absorption peaks for a target compound, plus one or morereference wavelengths to measure background absorption, can be projectedonto a liquid chamber containing a laminar flow with adjacent sampleliquid and reference liquids. The laser array is oriented such that thespots from the array run parallel to the flow of the liquid, and thenthe modulating scanning described herein scans these spots perpendicularto the fluid flow, and across any concentration gradient formed by theinterface between the sample and reference fluids. After interactingwith the fluid and being absorbed according to wavelength andconcentration, each of these spots is relayed to a correspondingAC-coupled infrared detector (in many cases part of an array, such as apyroelectric detector array). The modulation of each detector signalresulting from the modulating scanning corresponds to the differentialabsorption between reference and sample liquid at a particularwavelength; from these signals the concentration of one or morecompounds within the sample liquid may be calculated.

One embodiment of “modulation” scanning (i.e. scanning that is detectedby the AC detector module) may include rapid spatial scanning over smalldimensions (as may for example, be used to interrogate a particle in thefluid) and slower scans over larger dimensions as may be required tointerrogate the entire sample. Either scan may occur in 1 or 2dimensions. In one embodiment, a rapid 1-dimensional scan may be usedacross a particular interface or feature in the fluid where there is aconcentration gradient. A 2-dimensional scan may be used in a pattern tocover an area where there are concentration gradients or features on thescale of the entire sample. For example, a Lissajous-type scanningpattern may be used to relatively uniformly scan a 2D area of the sample(using simple control electronics).

Various beam or optical spot sizes and shapes may be used in thedisclosed technique to interrogate the sample. These include circularspots, but also elliptical spots, the latter particularly well-suitedfor 1-dimensional scanning perpendicular to the long axis of theelliptical spot. For example, when scanning over the interface betweentwo liquid flows in a flow chamber, an elliptical spot with a long axisparallel to the flow (and interface), and therefore perpendicular to thedirection of scanning of the beam over the sample (or sample past beam)may provide particularly high contrast as the spot moves over theinterface between liquids (compared to a more gradual change for acircular spot, for example) and may be used to detect opticalcharacteristics of the interface. Such a configuration would be validfor transmission, transflection, or surface-sampling opticalconfigurations such as ATR prisms integrated with the flow chamber.

FIG. 7 shows another embodiment of the disclosed technique; in thisinstance the sample chamber is scanned across the beam in order toinduce modulation according to gradients within the liquids. An infraredlaser source (SRC) 701 is collimated using lens 702, and focused ontothe sample chamber 705 using mirror 703 and lens 704. The sample isscanned using scanning subsystem (SCAN) 706, which could for example bea piezo transducer (1- or 2-axis) capable of scanning the sample at >1Hz, >10 Hz, >100 Hz or higher frequencies to achieve the signalmodulation described herein. A capturing lens 707 re-collimates thebeam, which is then focused onto detector subsystem (DET) 710 byfocusing lens 709. The signal from the detector subsystem may be used bya computer controller unit (COMP UNIT) 711 to calculate absorption andpossibly concentrations or other optical characteristics, which go tothe system output 712. This embodiment may have a drawback that thesample holder may have considerable mass and therefore require moreenergy to scan, and scanning may disturb the contents of the sampleholder. However, an advantage is that a very consistent optical spot ismaintained on the sample, reducing optical artifacts that result innon-signal modulation at the detector. In this embodiment, the sampleholder may be translated both by the scanning system, as well as asecondary translation system that allows the sample to be put in focus(i.e. “Z axis” scanning), and different portions of the sample may bemeasured.

FIG. 8 depicts an example sample holder for use in the disclosedtechnique. As light from laser sources in the mid-infrared is coherentand often has narrow bandwidth (monochromatic), issues of opticalinterference may become problematic. In the disclosed technique, whereone or more beams is scanned relative to the sample and sample holder,small changes in reflection from the interfaces of the sample holder,compounded by coherent light effects, may cause changes in intensity ofthe light at the detector that are not related to the sample itself; inaddition, optical interference effects within the sample holder maychange the effective optical power at the sample itself (standing waveeffects). Finally, reflections back to the laser source (opticalfeedback) may modulate the laser output (i.e. optical feedback). Oneembodiment may minimize changes in the optical path through the sampleholder, and minimize reflections from surfaces of this holder. Theexample shown in FIG. 8 consists of an infrared flow cell with surfaceangle at the Brewster angle, or the angle where p-polarized light istransmitted without reflection through surfaces. Mid-infrared light 801(shown here to be p-polarized) from a laser source (this is particularlytrue of QCLs) is highly polarized, and therefore this design may beemployed without significant losses or back-reflections. The examplesample holder shown in FIG. 8 consists of two infrared-transparentwindows 802 which appear on either side of a liquid sample channel 804,which may contain a stationary or flowing liquid sample. The thicknessof the windows 802 is for illustrative purposes only; typically thethickness of the windows will be many times the thickness of the liquidchamber or channel. The angle of incidence 803 from the surroundingmedium (typically air) into sample holder window surface is at theBrewster angle, where there is no reflection of p-polarized light;subsequent angles 805 (window-to-liquid) and 806 (window-to-air) as wellas the angle exiting the liquid into the window are all constructed,based on the respective refractive indices (at the operating wavelength)of the surrounding medium, window material, and liquid sample. In thismanner the transmitted light 807 is free of “ghost images” resultingfrom internal reflections, as well as free of “fringes” resulting fromresonant cavities inside the sample holder, or between the sample holderand other system components. This is of importance in the embodiment dueto the modulation scanning of the beam over the sample, and thereforethe sample holder. Such scanning may result in slight deviations ofincident angle, as well as scanning over slight thickness variationswithin the sample holder windows, and other path length variations, thatwould be amplified if resonant cavities were to form inside the sampleholder, or between the sample holder and other system components. In thepresent example, the beam would be scanned in and out of the plane ofthe paper relative to the sample holder (or, equivalently, the sampleholder is scanned), so as to keep the incident angles substantiallyidentical throughout the scan range.

Thus in one embodiment of the invention the fluid cell may comprise twooptically transmissive windows defining two surfaces of the fluidchannel, each window having a first surface in contact with the analyteor reference fluid and second surface, the angle of incidence of theoptical beam on the first and second surfaces substantially at theBrewster angle to reduce optical reflections relative to a non-Brewsterangle of incidence, and the optical beam being motion scanned in amanner to substantially maintain the Brewster angles at each samplinginterval of the detectors used in determining fluid opticalcharacteristics.

For semiconductor infrared laser sources such as QCLs, inherent spectrallinewidths, or width of individual lasing modes emitted from the laser,may be extremely narrow (<0.001 cm-1). As a result of these narrowlinewidths, resonant effects such as fringes may be very pronounced. Forsemiconductor-based laser sources in the infrared such as QCLs, it mayoften possible to “spread” the effective linewidth of the laser throughthe use of current modulation, which produces a rapid thermal modulationwithin the laser chip, and therefore refractive index changes thatresult in wavelength modulation (and concomitant amplitude modulation).In another embodiment, these lasers may be operated in pulsed mode,where their spectral linewidth may spread considerably. This isimportant because a broader linewidth reduces the coherence length ofthe emitted light—or the distance over which pronounced interferenceeffects may occur. In traditional infrared spectroscopy applicationswhere gases are measured, narrow linewidth is prized in order to makeprecise measurements of narrow gas absorption lines; however inliquid-phase samples, absorption peaks typically have peak widths on theorder of 5 cm-1 or more. As a result, embodiments of the disclosedtechnique may include modulation or pulsing of the laser light sourcesin order to reduce coherent artifacts within the system. The modulationof the laser source may be done at a higher frequency than themodulating scanning described herein, and may be done beyond thebandwidth of the primary detector used in the system. Significantthermal tuning (and therefore frequency broadening) can be achieved inQCL chips, for example, with modulating frequencies of 10-100 KHz, andeven 100-1000 KHz. Additionally, some QCL chips may be pulsed at highfrequency, for example 10-100 KHz and even higher. At these frequencies,thermal detectors such as pyroelectric detectors do not experience amodulated signal, but a DC average of this modulated or pulsed power,and therefore the dynamic range of the detector or associated circuitryis consumed by the modulation or pulsing.

FIGS. 9A and 9B show an example of a liquid chamber/channel-integratedattenuated total reflection (ATR) prism that could be integrated into anexample embodiment of the disclosed technique. This can be done toextend distances between components where back-reflections cannot beavoided to distances beyond the coherence length of the laser source(s).Such a configuration may be used in applications where the liquid mediumis highly absorptive (such as water, in large ranges of the mid-infraredrange), but narrow liquid channels that would allow sufficient lighttransmission are not feasible (because of the danger of clogging, forexample). Here a liquid channel 901 carrying a flow of liquid is shown;this channel is contained between two surfaces: top surface 902 whichneed not be transparent in the mid-infrared; and bottom surface 903which is constructed from an infrared-transmissive material, and has anintegrated ATR prism 904. Incoming infrared light 905 enters the prism(the light and entry surface may be oriented such that the entry is atthe Brewster angle, as described above), and then reflects one or moretimes from the surface in contact with the fluid sample. With each totalinternal reflection from this surface, there is some evanescentpenetration 906 of the light into the channel and therefore the sample,and absorption according to the wavelength, the chemical contents of thesample and their resonant infrared peaks. The exiting light 907 is thenrelayed to the AC detection subsystem as described above. In thisdesign, the beam and sample holder are scanned relative to one anotherin a direction perpendicular to the plane of the paper, such that theentry angle, reflection angles, and exit angles, as well as the internaldistances within the prism, remain identical. The front view of FIG. 9Bdepicts a cross-section from the direction in which the fluid flows,with two beams 908 showing the extremes of the scan range, and theliquid showing a concentration gradient within the range of this scanthat will result in a modulation signal at the detector, depending onthe incident laser wavelength. This configuration may be used, forexample, where a sample liquid is flowed in parallel with a referenceliquid, and the scanning beam is scanned back and forth across theinterface between these liquids. Any intensity modulation in thetransmitted light 907, then, indicates a differential in the contentsbetween sample and reference liquids—providing high detectionsensitivity at a frequency above low-frequency noise and system drifts.The example here, again, may be used where a transmission ortransflection measurement is not appropriate, because it is physicallydifficult to flow the sample liquid through a narrow enough channel (dueto viscosity, particulates that could cause clogs, etc.)

FIG. 10A shows an embodiment in which a scanned liquid sample includesdispersed solids or liquids—for example hydrocarbons dispersed in awater sample, or fat droplets in milk. Two incoming infrared beampositions (the extremes of a scan range) 1001 are shown as they aretransmitted through a liquid sample 1003 in a channel or chamber betweentwo infrared-transmissive windows 1002. In this embodiment, the liquidis shown to have two regions that the beam scan range straddles: onewithout (1003), and one with (1004) scattering particles such assuspended solids, suspended droplets, or other significant inclusionsother than dissolved chemicals. The gradient in such inclusions could bea result, for example, of two liquids in a laminar flow (in or out ofthe plane of the page)—one of which is a sample (typically the one withthe inclusions), and one of which is a reference liquid withoutinclusions, or with a known distribution of included particles ordroplets. As the beam passes through regions with these inclusions,light 1005 is scattered as a function of the size and shape of theinclusions, as well as the complex refractive index of the inclusionsrelative to the liquid medium carrying them. As described above,specific infrared wavelengths may be used where particular chemicalcomponents of the inclusions (or the medium) have sharp rises or dropsof refractive index (resonant regions), or high absorption. Thus, theability to measure scattered light as a function of wavelength can allowcalculation of various combinations of inclusion size, concentration,and chemical composition—or, may be used to calculate the concentrationof inclusions with a particular chemical makeup (for example, resonantMie scattering effects at specific wavelengths could be used to measureonly the concentration of droplets composed of hydrocarbons, vs gasbubbles or other inclusions in a liquid).

Particle when used generally herein may mean a particulate, droplet, gasbubble, undissolved analyte or other undissolved substance with achemical or optical characteristic different than the fluid that mostlysurrounds it within the fluidic channel. A particle or concentration ofmultiple particles will generally have a size or sizes that result inlight reflection, light scattering or measurable modulation oftransmission relative to fluid without such particle or particles. Sucha particle that is being transported or has been transported by thefluids, or adhering to a fluidic conduit side wall, whether in thechannel, or prior to or exiting from the fluid channel, may also beconsidered a particle unless specified otherwise. A particle may also bean analyte.

FIG. 10B shows an embodiment of the disclosed technique used to measuredispersed contents within the liquid sample of FIG. 10A. Light from aninfrared laser source 1007 (which, as in all examples herein, mayprovide multiple wavelengths, either sequentially or simultaneously) iscollimated by lens 1008 to provide a scanned beam 1001 to the liquidsample 1009. In this embodiment, scanning modulation is achieved byscanning the sample holder with the liquid sample back and forth acrossthe beam. Some light is transmitted directly 1006, with absorptionaccording to concentrations of species in the liquid sample andwavelength. Light that is scattered 1005 due to inclusions in the liquidemerges with a distribution of angles dependent upon the size andchemical composition of the inclusion. In this embodiment, a focusinglens 1010 is used to focus the directly-transmitted light through apinhole aperture 1011; this aperture transmits light that passeddirectly through the sample with only attenuation, but preferentiallyblocks light that has been scattered at an angle by inclusions in thesample. The light transmitted through the pinhole aperture is thendetected by a detector subsystem 1012 which is AC-coupled and designedto respond to signals at the frequency of the modulation scanning (ofthe sample holder past the beam). By measuring this signal as a functionof wavelength, it is then possible to calculate one or more ofcharacteristics of an analyte in the liquid, concentration ofinclusions, contents of these inclusions, and/or size of the inclusionsin the liquid sample.

In such scattering-measurement embodiments of the disclosed technique,it may be desirable to directly measure scattering; for example,inverting the spatial filter 1011 to block any directly-transmittedlight and measure only light scattered by the sample as it is scannedacross the beam. This may be repeated at several wavelengths in order tocalculate one or more of total concentration of an analyte in theliquid, concentration of inclusions, contents of these inclusions,and/or size of the inclusions in the liquid sample. In otherembodiments, largely directly-transmitted light may be separated fromlargely scattered light by use of mirrors and/or spatial filters andmeasured independently and simultaneously.

FIGS. 11A-11D are several graphs providing further explanation of ascattering measurement that may be employed in certain embodiments ofthe disclosed technique where particles, droplets or other inclusionsare dispersed in the liquid sample. For each graph, the horizontal axisis optical frequency, with higher frequencies (shorter wavelengths) tothe right of the graphs.

Graph 1101 shows the absorbance, as a function of wavenumber, of anexample compound, with a resonant absorption peak centered at va. Forstandard absorption measurements, a laser source may be configured toemit infrared light corresponding to this peak, and the beam scannedover the sample containing potential gradients in concentration of thispeak, resulting in a modulation of the transmitted light (as a result ofthe compound-specific absorption). Light at one or more otherwavelengths, typically nearby to the target absorption peak, may also beused to establish a “baseline” for the peak absorption measurement (i.e.cancel out other factors and overlapping absorption signatures—not shownhere).

Graph 1102 shows the real refractive index of the target compound (solidline) and liquid medium (dashed line) as a function of frequency. As aresult of the Kramers-Kronig relationship between real and complexrefractive index, the real index of the target compound displays a“wiggle” that is a derivative of the absorbance shown above it, inaddition to a constant term. In this example, the index of the medium isrelatively constant. As a result there is relatively rapid change (withfrequency) of index differential between the target compound and themedium, with a local maximum at vb and a local minimum at vc.

Graph 1103 illustrates the importance of this variation in indexdifferentials. This graph represents the scattering efficiency of adroplet or particle of the target compound resident in the medium. Thescattering is a function of the size of the inclusion (held constant forthe purpose of this illustration) vs the illuminating wavelength, aswell as the refractive index differential. There is a general upwardtrend towards higher frequencies (shorter wavelengths), as the size ofthe particle becomes larger compared to the wavelength. Superimposed onthis scattering “baseline” is the local variation due to the refractiveindex change around the resonant frequency of the compound (reallyspecific molecular bond vibration modes within the compound). Whereindex differential is higher (vb), scattering increases, and where it islower (vc), scattering decreases. This effect—resonant Miescattering—occurs over a short frequency range where other factors arerelatively constant.

As a result, in certain embodiments of the disclosed technique, asdescribed above, it is possible to measure compound-specific scatteringin a liquid sample. Substantially directly transmitted and scatteredlight may be measured separately, or the combined effects may bemeasured

Graph 1104 is a resulting extinction curve. In this compound signal, oneor more discrete frequency points may be used to measure thecharacteristics of the liquid with dissolved or dispersed components:frequencies v1 and v2 may be used to measure non-specific scatteringfrom the sample (and therefore indicate, generally, the level ofinclusions in the liquid); a laser at frequency va may be used to assessabsorption (at this frequency there is no net effect from resonant Miescattering, but includes the baseline Mie scatter) alone when baselinedusing non-resonant scattering measurements from v1 and v2. Finallymeasurements at frequencies vb and vc may be used to extract theresonant Mie scattering effect, and therefore compound-specificscattering by inclusions in the system. These measurements, made usingthe scanning modulation system described in the disclosed technique mayenable high accuracy calculation of dissolved and dispersed componentswithin a liquid sample.

The disclosed technique is primarily focused on the mid-infrared (2-20um) wavelength range where molecules have specific resonant absorptionfingerprints; furthermore the disclosed technique may be applied in theterahertz range (100-1000 um) to which infrared laser sources haverecently been extended, and where molecules likewise exhibitcharacteristic fingerprints. In this frequency range, it is alsopossible to measure interactions between molecules, or within molecules(such as proteins, when folding) using the spectroscopic techniquesdescribed herein. The disclosed technique may be used, for example, toscan the interface between two liquid samples that interact, providinghigh sensitivity to the resulting molecular interactions provided by thescanning-modulated liquid measurement system described herein. The lasermotion in the direction of stream flow further provides for ameasurement of the molecular interactions over time.

The disclosed technique comprises infrared and terahertz laser sourcesof all types—the key distinguishing features of such sources (as opposedto traditional incandescent or even synchrotron sources) being that:they provide relatively high power at specific wavelengths of interest;and they are coherent, small aperture sources that as a result may beefficiently collimated or focused onto a sample, and therefore providerelatively high optical power onto a limited area, which is then scannedto provide the modulation described herein. Specifically, quantumcascade lasers (QCLs) are a suitable source for many embodiments of thedisclosed technique, as they can be manufactured to emit light attailored wavelengths within the mid-infrared and terahertz bands thatare the subject of the disclosed technique. Furthermore, QCL sources maybe tunable (through the use of external gratings, tunable filters, orother mechanisms) over wavelength ranges suitable for measuring resonantabsorptions in liquid-phase samples; furthermore, monolithicallyintegrated arrays of QCLs with distinct wavelengths may be fabricated,again emitting over a range suitable for liquid-phase samplemeasurement. All of these types may be used in the disclosed technique.Other infrared laser sources, including CO2 lasers, lead-salt lasers,optical parametric oscillators, etc. may be used in the disclosedtechnique.

The disclosed technique may be used to measure impurities in liquids,for example hydrocarbons that may be present in water as a result ofhydrocarbon exploration, exploitation or processing operations. Anexample embodiment for this application comprises the following:

-   -   A mid-infrared QCL source configured to emit at a frequency        around a major hydrocarbon absorption band, for example 1460        cm-1. This QCL source is tunable such that it covers a range        that includes the hydrocarbon absorption band, but also adjacent        frequencies where hydrocarbons do not absorb as strongly (for        reference levels). This QCL source may be pulsed, or modulated        at high frequency (for example, 100 kHz) to spread its bandwidth        and avoid some coherent artifacts in the system.    -   A liquid handling system that introduces the liquid sample,        along with a reference liquid (pure water) into a flow chamber        (e.g. a channel of a microfluidic cell) where these liquids flow        in laminar fashion through a measurement cavity (e.g. an        interrogation region where the fluidics interact with the        radiation generated by the optical source).    -   One surface of this flow chamber is bordered by an        infrared-transparent window, for example CaF2 or ZnSe. This        window may have integrated into it an ATR prism which allows        multiple internal reflections of infrared light from the surface        in contact with the fluid chamber, these reflections occurring        along the axis of the flow.    -   Optical components to relay the infrared light from the QCL        source into the ATR prism, with the center position of the        reflections in the ATR being close to the interface of the        sample and reference liquid flows; the entry angle into the ATR        and the exit angle out of the ATR configured relative to the        polarization of the QCL source such that a minimum reflection        occurs at these surfaces, according to the Brewster angle        calculated using the index of the ATR prism material;    -   A sample scanning system that repetitively translates the sample        holder and ATR relative to the light source incident there upon,        and in a direction perpendicular to the flow and to the sequence        of reflections inside the ATR. This scanning system translates        the sample and the contained flow at roughly 100 Hz, for        example.    -   Optics to capture and relay the light emerging from the ATR        prism, which has evanescently interacted with the flow in the        chamber, to a detector subsystem;    -   A detector subsystem which is configured to detect the        transmitted infrared light, with electronics designed to isolate        and amplify the signal that results from the scanning of the        sample holder (and contained flow) and therefore the effect of        the hydrocarbon concentration gradient at the border between the        sample and reference liquid flows; further comprising a DC level        detector which measures the average power transmitted through        the system; for example, the AC detector in this system may be        based on a pyroelectric detector (which is inherently        AC-sensitive); the DC portion may be based on a thermopile        detector; both of these are uncooled, stable, broadband and        low-cost detectors;    -   A controller or computing system which:    -   tunes or switches the QCL source sequentially to wavelengths        corresponding to one or more reference wavelengths (where        hydrocarbon absorption is relatively weak) and peak absorption        wavelength (where hydrocarbons in question have relatively        strong absorption);    -   optionally controls the modulation scanning of the beam (i.e.        interrogation region) between the sample and reference flows,        within the ATR sample;    -   records the amplitude of modulation detected by the detector        subsystem, as well as the DC power level transmitted through the        system; normalizes the modulated power by the DC transmission;    -   calculates hydrocarbon concentration in the water by normalizing        signal at peak absorption wavelength by the signal at reference        wavelengths;    -   reports the hydrocarbon concentration in the sample;    -   optionally, controls the scanning or other translation mechanism        to position the fluid interface (between sample and reference)        at the center point of the beam vs sample holder scanning range;    -   optionally, occasionally positions the scanning range entirely        in the reference liquid, so as to extract a baseline signal        level where no concentration gradients are present;    -   optionally, stops all scanning motion and centers the beam on        the fluid interface to observe signal from any turbulence within        the flow, adjusting flow rates appropriately to achieve laminar        flow and therefore a clean interface between the two fluid        streams.    -   optionally calculates the hydrocarbon concentration using a        ratio of the detector signals for a reference and sample (i.e.        transmission), and the optical pathlength in the fluids

The example system enables the measurement of very low levels ofhydrocarbons dissolved in water samples through the use of the disclosedtechnique's unique infrared laser liquid-scanning plus AC detectionarchitecture.

The method of sample introduction into the microfluidic cell may beperformed in a system for online continuous measurements, or a samplefluid may be introduced into the system in “batch mode” whereby a staticvessel (e.g. plunger) is filled with the sample of interest, and thesample (and reference) fluids are introduced into the cell.

When measurements of emulsions, “dirty samples”, or samples that arelikely to leave contaminating residue in the cell are made, it ispossible to add a cleaner, which in one embodiment is opticallynon-interfering to the desired fluid characteristic measurement, to thereference and/or sample streams, such as a surfactant to removehydrophobic materials such as fats or oils, or by adding an appropriatesolvent. Alternatively, a cleaning solution may be periodicallyintroduced into the cell to flush the system and clean the cell. Thecleaning solution or another third reference sample may have very high,a known, or nominally 100% transmission to provide a measurement of thetotal laser power, thereby calibrating the prior relative amplitudemeasurement into a more accurate and calibrated absolute measurement.

In another embodiment, a series of measurement values are combined (e.g.co-added or averaged) to improve measurement sensitivity, and a likelypresence of a particle or bubble in the fluid channel is detectedthrough optical, pressure measurement, flow rate, fluid interfaceperturbation, change in the ratio of sample and reference detectoroutput signals, or other detection methods, and samples of the detectoroutput signal likely to have values perturbed by the particle or bubbleare excluded from the series of measurement values. The particle orbubble may be detected prior to entering the interrogation region or maybe detected in the interrogation region, and if detected prior toentering the interrogation region, the time the particle or bubbleenters the interrogation may be projected from the fluid or particlemotion (e.g. fluid velocity and distance between the detection point andthe interrogation region). The bubble or particle may not enter theinterrogation region and still have an effect on the dynamics of fluidmotion within the interrogation region (e.g. by effecting the motion ofthe fluid boundary), and thus values may still be excluded. The bubbleor particle may be swept along the channel or may be become lodged inthe cell channel or in the fluid paths or channels entering or exitingfrom the cell channel, and thus still effect the measurement value andrequire excluded values. The relative position of the interface regionbetween fluids and the interrogation region may be dynamically adjusted(e.g. offset from an operating position or a change in the averageposition during the motion modulation) during operation to account forthe presence of a detector or bubble in the channel.

One embodiment may include an analyzer with chamber or microfluidic cellthat (1) detects the presence of one or more analytes at a spectralwavelength where the one or more analytes have combined differentialabsorption relative to the solvent, and then (2) speciates between theanalytes and determines their concentration in the solution at one ormore other spectral wavelengths. One advantage of such a system is thatit may more rapidly detect at a single wavelength the totalconcentration of multiple analytes or more readily detect theconcentration of a single analyte in the absence of other analytes orinterfering substances. In one embodiment, an analyte bearing samplefluid is measured at a single wavelength and if the analyte absorptionexceeds a threshold, then the sample is held longer (i.e. literally heldlonger in the fluid channel or the analyzer prolongs a measurement of ananalyte sample relative the case where the threshold was not exceeded)and the tunable laser is used to measure absorption at additionalwavelengths in order to speciate the one or more analytes. It should berecognized that alternative combinations of wavelengths, solventabsorption and analyte absorption may be chosen for a particularapplication. In another embodiment, all of the analytes of interest haveabsorption at the selected wavelength that exceeds the solventabsorption and thus once again there is no combination of analyteconcentrations (other than none) where the combined analyte absorptionequals the solvent absorption.

The analyte and solvent may be immiscible liquids. It may beadvantageous to ensure formation of an emulsion of the analyte in thesolvent through means such as homogenization, shaking, addition of anemulsifier, or, by creating turbulent flow. The targeted particle sizemay be determined by the wavelength of laser light relative to theparticle size. Such a particle measurement system may be used to providefeedback into the means for emulsification. Such a feedback system mayalso be used to change the flow rates in the cell or the absorptionmeasurement time or both.

In various embodiments it may be advantageous to modulate the amplitudeor wavelength of the laser signal synchronously with the modulation ofthe interrogation region position relative to the fluids in the channel.For example, the laser signal may be turned off when the transitionregion is transiting the beam such that system power is conserved andabsorption measurements are only taken for the unmixed reference andsample streams.

FIG. 12 shows a first embodiment of “serial streaming” in which a liquidsample solution 10 containing an analyte of interest is introduced intoa fluid flow cell (or “flow cell”) 12 in either a continuous flowingstream, or in a flow-stop-measure-start-flow repeating sequence. In theflowing stream, a reference solution 14 (the order of sample andreference can be reversed) is introduced into the flow stream in such amanner as to create alternating segments or plugs in the flow stream ofsample 10 and reference 14 materials. These alternating segments areshown as S for sample and R for reference. A Mid-IR source 16, such as afixed frequency or tunable QCL laser 16 as shown, or one or more lasers,is tuned to a suitable wavelength for measuring the analyte(s) ofinterest, such as the peak of an absorbance feature chosen to minimizebackground interferences. The Mid-IR source 16 may be coupled to thefluid flow cell 12 through a fiber. The reference material is chosen asa suitable reference material or mixture representative of the samplebackground as previously disclosed. The reference may be inserted intothe sample stream using microfluidic techniques such as valves, mixers,or pumps (generally, flow-control devices), and/or the use of pressureto alternate the sample and reference streams, all as known in the art.In the illustrated example a switching valve 18 is employed.

FIGS. 13 and 14 illustrate an embodiment of “parallel streaming) inwhich a sampling beam interrogation region 220 (i.e. the region of theflow cell channel that transmits the laser beam and is the opticalabsorption detection region) is placed at or near the convergence of thesample and reference streams 222, 224 in a fluid channel where they areseparated by a fluid boundary or interface 226 as previously disclosedfor laminar flow streams. In these Figures the laser beam is incidentorthogonal to the page. By varying the relative pressure of the twostreams 222, 224, the widths of the streams 222, 224 in the fluidchannel 228 can be varied such that the laser beam alternatively passesthrough one stream 224 (FIG. 13) and then the other 222 (FIG. 14). Bytime varying the pressure differential between the two streams 222, 224,the frequency or rate of measurement of the sample and reference may bevaried, as can the transition time when regions of both the reference224 and sample 222 are within the interrogation region 220. The size ofthe interrogation region 220, which is substantially the same as thelaser beam diameter, may be less than the width of the individualstreams 222, 224, enabling a discrete sampling of each stream 222, 224.Alternatively, the laser beam diameter may exceed the width of theindividual streams 222, 224, and multiple detectors may be used tospatially sample across the transition region or across the sample,reference and transition region streams.

Thus three general types of embodiments are contemplated for performingsample characteristic measurements as described: motion of the opticalbeam relative to the fluid channel, motion of the fluid in a serialstreaming manner in the channel, and motion of the fluid in a parallelstreaming manner in the channel. Combinations of these techniques arepossible, as is the general use of laser or fluid motion in order totranslate the position of interrogation point relative the microfluidiccell or fluid boundary for subsequent or simultaneous sample-referencedifferential measurements in accordance with the invention.

Thus, one embodiment may include:

an optical source and an optical detector defining a beam path of anoptical beam;

a fluid flow cell disposed on the beam path defining an interrogationregion in a fluid channel in the fluid flow cell in which the opticalbeam interacts with a fluid;

one or more flow-control devices configured to conduct an analyte fluidand a reference fluid stream through the fluid channel, a fluid boundaryregion separating the analyte and reference fluids when flowing togetherthrough the fluid channel;

a controller operative (1) to sample an output signal from a transducerto detect a particle within the fluid channel, (2) to generate a motionmodulation signal having a time-varying characteristic to cause theparticle to be moved relative to the interrogation region (3) to samplethe output of the optical detector at one interval of the motionmodulation signal during which the interrogation region containssubstantially the particle and at a second interval during which theinterrogation region contains substantially the fluid surrounding theparticle, thereby generating corresponding output signal samples, and(3) to determine from the output signal samples a measurement valueindicative of an optically measured characteristic of the particle.

The embodiment may further include a motion control device configured toposition the interrogation region location in the fluid channel; anoptical signal incident on the detector that has been spatially filteredfor the purpose of detecting scattered optical signal, an optical signalincident on the detector that has been spatially filtered for thepurpose of removing scattered optical signal; an optically measuredcharacteristic of the particle that is an optically measuredcharacteristic of an interaction of the particle and the surroundingfluid; an optically measured characteristic of the particle that is anoptically measured characteristic of an interaction of the particle andthe optical beam; or an optically measured characteristic of theparticle that is an optically measured characteristic of an interactionof the particle and the microfluidic cell.

The transducer used to detect the particle in the embodiment may theoptical detector or may be another transducer type. For example, thetransducer may be a visible imager viewing the channel that provides anoutput signal to the controller indicative of the presence of theparticle and its position in time in the channel.

FIG. 15 shows an embodiment in which the a sequence of samples areprovided by sampling tubes 241 to a fluidic laser-based analyzer 240,which may be realized according to one of the above-describedembodiments. In particular, the sequence is obtained by sampling astream 242 at multiple sampling points, which may be spatially dispersedin such a manner as to ensure that the collected samples arerepresentative of the liquid within the object being sampled (e.g. waterin a pipe or container). In this manner, the small volume of themicrofluidic transmission cell can efficiently sample the liquid in alarger object. Samples are sequenced by sequential operation of samplingvalves 244. The spatial positioning of the sampling points may bedetermined by the time variant nature of the liquid in the sampledobject (e.g. flowing water in a pipeline) and the timing of theabsorption measurement. For example, as shown in FIG. 15, samplingpoints may be dispersed in the direction of water flow such that thesamples are measured in the cell as if they were collectedsimultaneously in a plane that is perpendicular to the direction ofwater flow. In another embodiment, each of the sample points may feedseparate sampling cells each with their own laser and detector, orsharing the same laser using beam splitting techniques well known toexperts in the art. In another embodiment, each of the samples are mixedto create an “average” sample prior to being measured by absorption. Inanother embodiment, samples collected by each of the sampling points aresequentially passed under the measurement window permitting the separateabsorption measurement of each sampling point. The separate absorptionpoints may then be averaged in signal processing electronics. In anotherembodiment, the multiple samples may be combined in a high streamvelocity channel that is then sampled by the flow cell at a lower streamvelocity. In another embodiment, the reference fluid may be extractedfrom sampling stream 242 at a different location than the samples 241and thus provide for a measurement of the change over time in flowingstream.

Sample cell or feed lines or both can be temperature controlled to bringthe fluids to well controlled, constant temperature before beingmeasured in the interrogation region. Accurate temperature controlalleviates temperature dependent spectral changes (very common in polarsolvents such as water, very problematic in milk measurements).

In some measurement applications it may be necessary to characterizesmall volumes of analyte fluids. The analyte fluid volume may on theorder of the volume of the fluid channel and may even approach thevolume of the interrogation region in the channel. Some embodiments ofthe subject invention may incorporate techniques for managing andmeasurement of small analyte sample volumes. An analyte sample may beinserted as a one or more fluid plugs in a serial stream that is flowedthrough the cell channel, each plug or plugs surrounded by referencefluid. Thus the serial stream of reference and analyte fluids volumesmay not be symmetrical but may have larger plugs of reference thananalyte fluids and the sample controller may generate a sampling signalwhen the sample fluid is in the interrogation region. The analyte samplemay be directly injected into the incoming streams or cell channel usinginjection syringes, PZT pistons, “T” fluidic junctions, or similarapproaches as known in the art, to create an analyte sample plug that ismeasured in the interrogation region and pushed through and out of thecell channel by the reference fluid, the analyte optical characteristicsdetermine by the measurement of sample and reference fluids aspreviously disclosed. In one embodiment, the sample may be dropped intoa fluidic cell channel with only three sides (i.e. with a pipette or eyedropper) and then the fourth side (e.g. a “lid”) inserted to define aclosed fluidic channel with inlet and outlet channels. The insertablefourth side of the channel may be a window of the cell through which theoptical beam is passed or may be a side of the channel that does notdefine the optical path length of the optical beam in the interrogationregion.

The timing of injection and fluid flow rates, and the volume of theanalyte sample, may be determined based on the rate of fluid boundaryregion interaction (e.g. diffusion) such that the desired analyte orboundary region optical characteristic measurement is achieved.Specifically the controller timing signals may include sampling by thedetector of a stationary analyte fluid optical signal, the initiation offluid streaming whereby the analyte sample is replaced in theinterrogation region by the reference fluid, and sampling by thedetector of the stationary or moving reference fluid optical signal. Thereference in these embodiments for measuring small analyte samplevolumes may be a gas rather than a liquid. Thus one embodiment of thesubject invention may the measurement of a stationary plug of analytesample or reference in a serial stream where the volume of the analytesample is materially less than the volume of the reference fluid thatprecedes or follows it in the serial stream, and the system controllergenerates timing signals for sampling of the detector signal whenanalyte sample and reference are in the interrogation region, and formoving the fluid stream in the channel to replace the analyte sample orreference with the other in the interrogation region.

The length and relative spatial positioning of the microfluidic streamchannels may be determined in part to ensure the desired temperatureuniformity of the solutions being tested. The microfluidic cell mayinclude a heater (or be mounted on a thermoelectric cooler) andtemperature sensor to control the temperature of the cell and therebythe solutions flowing in the cell. The temperature sensor may be used toprovide a reading of the stream temperature for use in determining fluidcharacteristics (e.g. absorption) by enabling an adjustment in thecalculated characteristic due to temperature.

The absorption of light by many liquids has a dependence on temperature.In one embodiment, the temperature of the reference or signal liquids inthe cell are changed in a controlled manner over time in order tomeasure an optical characteristic of the fluids or to provide areference or calibration signal. For example, the “gain” of the systemmay be determined by measuring the known absorption of the referenceliquid at two different reference liquid temperatures.

Variable temperature control of the cell can also be used to study theeffects of temperature on chemical and biological systems. For exampleby incorporation of a variable temperature controlled cell one can studythe conformational changes of a protein or the rate of a chemicalreaction. Many chemical and biological molecules undergo rapid changesas a function of temperature. For examples, proteins undergo rapidconformational changes as a function of temperature. Due to the smallvolume of fluid typically passing through the microfluidic channel, itis well known that the fluid comes to rapid equilibrium with the cell.This allows for the possibility of measuring the effects of rapidtemperature change by introducing the sample and reference fluids intothe cell at a higher or lower temperature then the cell, and measuringthe fluids at multiple spatial positions within the channel whichcorrespond to different periods of time that the fluids have been in thecell. This could allow probing the kinetics of the chemical orbiological system. Other embodiments may modulate the fluids within thechannel by changing the microfluidic cell temperature or the use ofdiscrete heaters for changing the temperature of the individual fluidsseparately.

In another embodiment, a laser may be used to temperature modulate thefluids prior to or simultaneous with the measurement of a fluidcharacteristic. The modulation may be at constant laser power (i.e. thelaser increases the fluid temperature through absorption) or the laserpower may be frequency modulated where the frequency may be a frequencythat is less than or greater than the alternating frequency of referenceand sample measurement.

FIG. 16 illustrates an embodiment that includes an “imager” including asecond source 260 and imaging detector 262 to simultaneously view thestream passing through the interrogation laser beam 264 at a secondwavelength of operation. In this embodiment the sampling cell windowsare substantially transmissive at both the laser and imager wavelengths.The imager may include magnification optics and use a shorter wavelengththan the laser 266 (i.e. visible imager and IR laser 266). The signalsfrom the imaging detector 262 and laser detector 268 pass to signalprocessing electronics (not shown), which uses the information in theimaging channel to improve the sensitivity or resolution of theabsorption measurement. For example, a second channel imaging device maylook for regions in the stream with particular characteristics (i.e.free of particulates or emulsions, a desired emulsion characteristics,flow rate, presence of a particle, etc.) and gate the measurement of theabsorption to be inside of or outside of this region. The second channeldevice may be used to look for effects induced by the laser beam in thestream, such as laser induced fluorescence or thermal effects due toheating of the liquid stream. The second channel device may be used tomonitor the cell channel window transmission for maintenance purposes,such as determining when the cell should be cleaned or replaced. Thesecond channel device may be used to quantify particulates or otherobjects within the stream. The second channel optical signal may beenhanced by the use of an optical source such as an illuminator, LED orlaser.

It may be possible to extract analyte diffusivities or other fluidboundary characteristics simultaneously during spectroscopymeasurements. For example, during modulation at the analyte-referencefluid interface, the spectroscopy instrumentation is measuring analyteconcentration at a fixed position along the channel (e.g. at theinterrogation region) or analogously a fixed time after the fluidstreams meet. Considering that the interrogation beam is modulatedacross the in interface, the measured analyte concentration signal overtime is effectively a spatial concentration profile of the analyte. Theconcentration profile depends upon the diffusion coefficient, which canbe ascertained directly from the concentration profile itself, given thediffusion time. The diameter of the interrogation beam may be reduced toimprove measurement accuracy. For example, the beam diameter may be onetenth of the expected width of the inter-diffusion at the interrogationregion. As disclosed with respect to multiple interrogation regions, theposition of the interrogation region along the fluid channel (i.e. toselect longer or shorter times of inter-diffusion at constant streamvelocity from the point of initial stream contact) may be varied. Inother embodiments, the fluid velocity may be varied to change the timeof fluid interaction (e.g. inter-diffusion) prior to arrival at theinterrogation region.

Thus one embodiment of the invention may include:

an optical source and an optical detector defining a beam path of anoptical beam;

a fluid flow cell disposed on the beam path defining an interrogationregion in a fluid channel in the fluid flow cell in which the opticalbeam interacts with a fluid;

an integrated temperature controller to provide constant or variabletemperature control of the cell;

one or more flow-control devices configured to conduct an analyte fluidand a reference fluid stream through the fluid channel, a fluid boundaryregion separating the analyte and reference fluids when flowing togetherthrough the fluid channel;

a controller operative (1) to generate a motion modulation signal havinga time-varying characteristic to cause the interrogation region to bemoved relative to the fluid boundary accordingly, (2) to sample anoutput signal from the optical detector at one interval of the motionmodulation signal during which the interrogation region contains moreboundary region fluid than analyte and reference fluid and at a secondinterval during which the interrogation region contains more analyte orreference fluid than boundary region fluid, thereby generatingcorresponding output signal samples, and (3) to determine from theoutput signal samples a measurement value indicative of an opticallymeasured characteristic of the interaction of the analyte and referencefluids.

The embodiment may further include a motion control device configured toposition the interrogation region at two or more positions in the fluidchannel in the direction of fluid flow where the optically measuredcharacteristic is determined at each interrogation region position andused to determine a variation in time of the optically measuredcharacteristic. The embodiment may further include one or morestructures within the optical channel that modify the mixing of theanalyte fluid and reference fluid in the fluid boundary region, and eachsuch structure may be measured by the analyzer to determine an opticalcharacteristic resulting from the different levels of fluidic mixing.

Bubbles, particulates, undissolved analytes and other objects in thestream may interfere with the flow of liquid in the cell. Particulatesof higher density fluids may be added to the streams and the streamsoperated at an increased Reynolds number relative to non-cleaningoperation to dislodge or remove the objects from the cell, and suchparticulates may be introduced as part of a special “cleaning stream” oras dispersed particles in the analyte and/or reference streams wheremeasurements are performed between the particles. Stream velocity may beincreased periodically to perform the same function. A laser opticalsource may also be used to heat the objects in order to dissipate,dislodge or remove the objects from the interrogation region. The lasermay also be pointed or translated (or the cell translated relative thelaser) in order to perform the same function at locations other than theinterrogation region.

The detector may have an optical filter to pass light from the opticalsource and block light at other wavelengths, as for example, may beemitted through blackbody emission from objects or liquids heated by theoptical source.

When objects in the streams have an optical absorption greater or lessthan the liquids in which they are contained, differential heating mayoccur. For example, an oil droplet may be heated above the temperatureof the water in an oil-in-water measurement where some of the oil may beimmiscible. Through blackbody emission, this differential signal may beobservable when collected by an infrared point or imaging detector. Theinfrared detector may have an optical filter to screen out the emissionfrom the optical source. The optical filter may also be a bandpassfilter designed to pass light at specific wavelengths where the liquidhas higher optical transmission. In this manner an optical source at onewavelength may be used to differentially heat an object in the liquid tocreate a differential optical signal between liquid and object throughblackbody emission that is then collected by a filter and detector at awavelength different than the optical source. Thus, in one embodimentboth transmission and emission detection may be performed, wheretransmission is used to detect immiscible analytes and infrared emissionis used to detect or image non-immiscible analytes. The emissionmeasurement may be taken on one side of the cell (e.g. same side as theoptical source) to minimize absorption in the liquid (i.e. shorterpathlength) with the transmission measurement taken on the oppositesurface to achieve full transmission through the cell.

FIGS. 17-18 show an embodiment making use of one (or optionally more)PZT piston type side injection pumps 300 (or equivalent as known in theart) in the region after the two streams have merged to move thereference and analyte streams through the interrogation area. The PZTpump may be advantageous because it may not be a pump in the traditionalsense of injecting liquid; it could just displace a volume in anoscillatory manner (e.g. by deforming a compliant microchannel substrateor through the use of a piezoelectric diaphragm micro-pump as known inthe industry) to move the stream boundary. The use of PZT pumps and thelocation of the pumps may be selected to provide an increased speed ofoscillation relative to modulation of the reference and analyte throughthe interrogation point using pressure differentials of the referenceand analyte streams prior to merging (as described previously). Othertypes of pumps may be used. In one embodiment, the oscillations may takeplace at a 1 kHz rate. A lens or aperture may be used to create asmaller interrogation area. The sampling cell and fluid channel may bedesigned to support multiple lateral “side to side” (e.g. right to leftto right in FIGS. 17-18) oscillations of the fluid boundary over thelength of the fluid channel.

FIG. 19 shows an embodiment in which the PZT pump may be used as theinjection point for the analyte B into a reference liquid A (theconverse also being possible). The velocity of the reference stream A,and physical dimensions of the PZT injection orifice, the PZT pumppressure and modulation frequency may all be optimized to achieve acertain characteristic of the interrogation point, such as its physicaldimensions. In one embodiment the reference liquid A may not bestreaming and may be stationary for the measurement period, and thenflushed to remove liquid A and analyte B. The time between flushes maybe a function of the rate of diffusion of the analyte into the referenceliquid. It may also be related to the viscoelastic and surface adhesionproperties.

Note that as described above for other embodiments, the fluid boundaryregion between the reference and analyte streams may be predominatelycontinuous in nature as in parallel-streaming, that is the boundaryregion is in the general direction of the stream flow, is modulated in adirection orthogonal to the direction of fluid flow (i.e. across thechannel rather than along the channel) and is present in a cross sectionof the liquid channel (i.e. a 2-D slice taken into page as shown inFIGS. 17-18) over at least the period or time of the measurement, withthe boundary region substantially or an average parallel (discountingno-slip boundaries proximate to surfaces) to the direction of fluid flowwhen traveling through the fluid channel. The fluid boundary region mayalso be discontinuous in nature (discounting no-slip boundariesproximate to surfaces), that is the boundary may be substantially or onaverage orthogonal to the direction of stream flow and traveling in thedirection of fluid flow as may be in serial-streaming, and thus one ormore boundary transitions may cross the interrogation region during themeasurement or between measurements (i.e. the boundary region betweenfluids A and B crosses the interrogation region periodically with anorientation mostly orthogonal to the velocity vector of the fluid flow,ignoring the effect of any no-slip boundary condition). The boundaryregion may also be created where there is not continuous flow of liquidthrough the interrogation region as shown in FIG. 19 except with fluid Astationary for the period of an analyte measurement sample.

Heating of the liquid through optical absorption may result in a changein the optical transmission due to the temperature coefficient ofabsorptivity. If the reference streams are not identical in flowcharacteristics within the interrogation region, heating may result in adifference in transmission between reference and sample due solely todifferential thermal heating. In one embodiment, the position of theinterrogation region within the channel is adjusted to minimize (or nullout) the difference in transmission. This may be a factory or fieldcorrection. In another embodiment, the differential flow characteristicsmay be adjusted through, for example changing the differential pressurebetween reference and sample to achieve the same null condition in theabsence of the analyte of interest (i.e. the null is achieved for areference versus reference condition).

In the same manner, the other stream asymmetries may be adjusted toachieve a desired null or non-zero differential transmission betweenreference and sample streams. For example, differences in refractiveindex between sample and reference may vary the effective signalcollected by the detector, and the flow characteristics may be adjustedto achieve the desired differential detector signal.

In another embodiment, a heater in close proximity to one of the inletchannels may be used to differentially heat reference or sample stream.The differential heating may be used to null or reduced an opticallyinduced differential signal in the interrogation region.

In another embodiment, the optical power may be adjusted as a functionof optical wavelength. The optical power may be measured by a detector(i.e. through the use of a beam splitter to tap off part of the opticalbeam) and the laser operating parameters adjusted to maintain constantpower as a function of optical beam wavelength in a tunable wavelengthoptical source. The optical power may be varied to “null”thermo-optically induced differential signals.

The channel may also be designed to have different dimensions (e.g.optical transmission pathlengths) at different locations, and thus a twodimensional moveable interrogation region may be advantageous, forexample, to change the optical pathlength through the fluids. This mayextend the dynamic range of the measurement to accommodate differentanalyte concentrations or different fluid absorption. The systemcontroller may have a method of searching for the best interrogationregion location in the channel for taking the measurement of interestthat includes taking a measurement, moving the interrogation regions inthe fluid channel, taking another measurement, calculating the bettermeasurement point, and then taking a series of measurements at apreferred measurement point. The measurement of interest may be or maybe related to the signal level on the detector, the measurement signalto noise ratio or the optical absorption of the reference or analytefluids. The optical measurement point may be one that provides a ratioof reference and sample intensities equal to a desired value (e.g. 1, asfor example would be the target value when reference and sample have thesame absorption at the interrogation wavelength). In one embodiment, theposition, size or shape of the interrogation region in the fluid channelmay be adjusted in a feedback loop over more than one fluid modulationsignal period (i.e. a cycle of one signal and reference measurement) toset the measured optical characteristic at a desired value forsubsequent operation of the fluid analyzer. The size or shape of theinterrogation region may be changed through the use of lenses or otheroptical elements, or by spatial movement of the fluid cell and opticalsource relative to each other or relative to other optical elementswithin the analyzer.

FIG. 20 illustrates an alternative configuration that can be used forincreased measurement sensitivity in fluids with less absorption of theoptical beam and thus may prefer increased pathlengths. The increasedpathlength may be achieved by directing the moving optical beam in thedirection of fluid flow down the channel 320, rather than across thechannel substantially across the direction of fluid flow. Theinterrogation area 324 may cross a boundary region between streams A andB at one of the beam entrances or exits from the channel. Thus theinterrogation area may primarily be in the middle of the channel in thedirection of fluid flow to avoid sampling of no-slip regions on thechannel sides and thereby improve measurement system sensitivity.

A reference solution may be used in both the reference and samplestreams in order to provide a “zero” point or other calibration of thesystem.

In another embodiment, the reference fluid may be extracted from thesame source as the sample fluid except at a different point in time,that is the reference fluid may be a time delayed version of the samplefluid (or vice versa). This embodiment may be advantageous in detectingchanges in fluids or solutions over time, as may occur, by way ofexample, due to the introduction of contaminants, or the result of achemical or biological process that evolves over time. The change overtime may be accumulated or integrated to show not only a change overindividual increments of time but the total change from the start of theprocess that is the accumulation or sum of the change over multipleincrements (e.g. from the time the process was first sampled). Thereference fluid may also be the analyte fluid with the analyte to bemeasured by the fluid analyzer removed by filtering, chemical processesor other means.

In one embodiment, the analyzer may be designed to operate “in situ”where the microfluidic cell is proximate or internal to the sample beingmeasured (e.g. within a process reactor vessel), such as may beperformed during the fermentation or purification processes of biologicmanufacturing. During in situ operations, it may be important to ensurethat the measurement itself does not have a material impact ordiscernable effect on the process. Thus, the analyzer may be designed tosample fluid volumes that are much less than the volume of liquid in theprocess reactor. The analyzer may be designed to return the sampledliquid back to the process reactor. The reference fluid may be a timedelayed version of the sample fluid which may or may not be returned tothe reactor vessel. The reference fluid may be an external fluidselected to not materially affect the reactor process if streamed intothe vessel after measurement. The reference fluid may be filtered orchemically altered version of the analyte fluid where an analyte (e.g.protein) to be measured has been removed from, or chemically altered in,the liquid. The reference fluid may be a fluid necessary for the processreaction and thus is streamed into the process reactor following themeasurement.

The presence of an analyte in the sample fluid may displace some volumeof the analyte sample fluid. Since both the analyte and fluid may absorbat the interrogation wavelength(s), the signal on the detector for theanalyte sample measurement may be the combined absorption of the analyteand the undisplaced fluid. If the reference fluid is the analyte samplefluid without the analyte, then a combined analyte sample and referencedetermination of the analyte optical characteristic in accordance withthe invention may include an “offset” term resulting from the absorptioncharacteristics of the displaced fluid, which will be different than theanalyte to be measured. In one embodiment of the invention, a knownoptical characteristic of the reference fluid (e.g. spectral absorption)may be used to calculate a correction to a measurement of an analyteoptical characteristic to account the displacement of the sample fluidby the sample analyte.

In one embodiment, the fluid analyzer may include a feedback systemincluding a measurement system for detecting at least one spatiallocation of the boundary region within the fluidic channel, the locationand motion of a particle within a fluid, or some other fluidic parameteror optical characteristic, a signal processor (e.g. controller) forusing the detection to calculate a new operating parameter of themeasurement system, and a control system for changing the operatingparameter. Measurement system operating parameters or characteristicsthat may include: fluid velocity; fluid Reynolds number; fluid pressure;a fluid channel dimension, orifice or valve; optical beam power; opticalbeam or interrogation region location; interrogation region crosssectional area or volume; volume or ppm of cleaning fluid; interrogationregion location in a channel; timing of the fluid modulation; timing ofa transducer signal acquisition; timing of the optical beam wavelength,power or frequency variations; amount of optical beam focusing withinthe interrogation region; transducer location relative to the cell;choice of transducer in an array of transducers for use in calculatingthe analyte property of interest; selection of light source; selectionof inlet or outlet channel; cell or channel temperature; power to anelement for controlling the microfluidic cell or individual channeltemperature; the frequency of calibration using a known input fluid oranalyte property; phase of a coherent optical beam; optical fringesincident on the transducer; selection of optical pathlength in amultiple pathlength cell; interaction time or other characteristics atthe fluid interfaces or particle-fluid interfaces; optical absorption ofthe analyte or reference fluids; phase of the fluid modulation relativeto the transducer signal integration period; volume of liquid in aserial streaming packet; amount of contamination on a channel surface;amount of optical power incident on the cell that is not transmittedthrough the interrogation region to the transducer due to refection orother means other than analyte absorption; stroke length of a 1-D pump;motion of a flexible membrane.

In one embodiment, the analyte fluid and the reference fluid may bechosen to have substantially the same value for an optically measuredcharacteristic and an operating condition of the fluid analyzer isadjusted in a feedback loop over more than operating condition signalperiod to set the measurement value at a desired level for subsequentoperation of the fluid analyzer. The operating condition signal may begenerated by a controller and one form of the operating condition signalmay a periodic motion modulation signal used to control the fluid orinterrogation region positions in the fluid channel.

In one embodiment, the effective path length may be determined with anabsorption measurement of a fluid with a known analyte concentration,and the effective pathlength used in the signal processing to determinean unknown analyte concentration. The pathlength may be measured byinsertion of an analyte X1 of known concentration C1 into the analyte orreference fluid and measurement of the absorption at a wavelength Y1other than the desired absorption wavelength YU of an unknown analyte XUwith unknown concentration CU. Analytes XU and X1 may have a largemeasured differential absorption difference at both Y1 and YU.

An analyte X2 of known concentration C2 may be inserted into a firstanalyte fluid for determining the effective pathlength at wavelength Y1(e.g. at the optimal wavelength for XU), without the presence of XU(note: X2 may be XU of known concentration). The pathlength thusdetermined may be used in determining the pathlength in a measurement ofCU. Thus, more generally, the analyte or reference fluid may contain ananalyte X1 with a known value C1 for the optical characteristic, and themeasured value of C1 and the actual known value of C1 are used indetermining (e.g. through a calculated effective pathlength) themeasured value of an analyte XU in the analyte fluid with an unknownvalue of the optical characteristic CU.

Thus, more generally, in one embodiment of the present invention theanalyte fluid or reference fluid may contain an analyte with a knownvalue of an optically measured characteristic, and (1) measurement valueof an analyte or reference fluid optical characteristic and (2) theknown value for of an optically measured characteristic are used indetermining the optically measured characteristic of the analyte fluid.

In applications such as sensing oil in water, the oil (i.e. analyte) mayadhere to and contaminate the optical surfaces of the cell over time.One advantage of the referencing techniques as described herein is thatsuch static contaminate is present for both reference and samplemeasurement and thus can be referenced out of the measurement, as forexample by the taking the ratio of the two measurements. During themeasurement of the reference, a measurement of the change in referencestream transmission over time may be used to determine when windowcontaminate reaches a threshold value requiring cleaning or flushing ofthe cell interior surfaces (e.g. a detergent flush). Measurement of thechange in transmission may include measurement of the emitted laserpower through the use of an independent laser power measuring detector.Measurement of the change in transmission may include measurement of theabsorption when both reference and sample are the same fluid.Measurement of multiple wavelengths with a tunable laser may also beused to determine the level of cell contamination, type of specificcontaminating analyte or both.

Described herein is a method of measuring an analyte (e.g. oil orcontaminant) in a liquid with one or more of the following:

-   -   1. Creating adjacent spatial regions of a reference liquid and        an analyte liquid (e.g. water reference and oil-in-water as        analyte liquid, the reference and liquid being the same fluids        except for the presence of the oil)    -   2. Illuminating an interrogation region with a light source at        one or more optical wavelengths (e.g. a wavelength of infrared        laser light)    -   3. Moving the interrogation region between the reference and        analyte liquids such that the interrogation region contains        predominately the reference liquid and then the analyte liquid    -   4. Measuring a resultant time varying interrogation signal with        a transducer (e.g. an infrared detector measuring modulated        transmitted or reflected light)    -   5. Using control electronics to process the time varying        transducer signal over one or more cycles of the interrogation        signal to calculate a desired analyte optical characteristic        (e.g. the amount in ppm of the analyte oil in the analyte liquid        water as a form of spectroscopy)

The method may additionally be including one or more of the following:

-   -   1. Modulating the interrogation light source to improve analyte        detection sensitivity    -   2. Adding multiple interrogation regions, each with their own        illumination source at a different spectral wavelength or        different transducer for measuring physical properties    -   3. Adding multiple interrogation regions, each with the same        illumination source    -   4. Measuring the optical thickness of the reference liquid or        analyte liquid at the interrogation region    -   a. Using signal processing, simultaneously or sequentially, to        correct the analyte property being measured to account for        variations in optical thickness of the reference liquid, the        analyte liquid or both.    -   5. Moving the boundary region between fluids by varying the        pressure of the reference liquid, analyte liquid or both    -   a. Generating the pressure variation through the use of a valve        or a pump or both where the boundary region moves simultaneously        or synchronously or both with the pressure variation at the pump        or valve.    -   6. Modulating the light source in intensity or wavelength (or        both), and using that modulation signal from the electronics to        improve the accuracy of measurement of the analyte property.

Overall, the disclosed technique may employ four types of motion:

-   -   1) Laser motion scanning of the laser beam (and therefor the        interrogation region) relative to the fluid channel and while        performing a sampling of sample, reference or interface region        as part of a measurement that combines at least two of the        measurements to determine a characteristic of the fluid.    -   2) Fluidic motion scanning of the fluids (and fluidic interface        region) at an interrogation point that is fixed relative to the        channel while performing a sampling of sample, reference or        interface region as part of a measurement that combines at least        two of the measurements to determine a characteristic of the        fluid.    -   3) Laser motion translation of the laser beam relative to the        fluid channel in order to determine a location in the channel        for motion scanning of the laser beam or fluids.    -   4) Fluidic motion translation of the fluids and fluidic boundary        in order to determine a location in the channel for motion        scanning of the laser beam or fluids.

The embodiments as disclosed herein may use one or more of these typesof motion. For example, a particulate in the channel may be laser motionscanned by moving the interrogation region with respect to a particle,or may be fluidic motion scanned by moving the fluid and therefor aparticle contained therein with respect to a fixed interrogation regionin the channel. Similarly, a particle traveling down the channel in afluid stream may be fluid motion scanned while the interrogation regionis translated down the channel through laser motion translation.

In one embodiment, laser and fluidic motion scanning and translation maybe performed simultaneously. The timing of a first motion waveform (e.g.a sinusoidal signal) that determines the laser motion (the laser orinterrogation region motion or motion modulation waveform) and a secondmotion waveform (the fluidic motion or motion modulation waveform) thatdetermines the fluidic motion may be synchronous or asynchronous withrespect to each other. The various translation and scanning laser andfluidic waveforms may be correlated or superimposed with respect to eachother, and the actual spatial motions of the laser and the fluidicboundary thus may be correlated with respect to each, correlationimplying that the waveforms or motions are not independent of eachother. For example, one or more of the following operating scenarios mayperformed by an analyzer system:

-   -   Synchronous and out of phase laser motion scanning and parallel        streaming fluidic motion scanning such that the effective        velocity of the interrogation region with respect to the fluid        is increased over the effective velocity for either laser motion        scanning or fluidic motion scanning alone (e.g. the optical beam        is moved in the opposite direction to the direction of motion of        the parallel streaming fluid boundary during the sampling of the        streams). The fluidic motion and the interrogation region motion        waveforms may be at the same frequency or an integer multiple of        frequencies with respect to each other.    -   Synchronous and in phase laser motion scanning and parallel        streaming fluidic motion scanning such that the effective        velocity of the optical beam and interrogation region with        respect to the fluid is decreased over the effective velocity        for either laser motion scanning or fluidic motion scanning        alone (e.g. the optical beam is moved in the same direction as        the direction of motion of the parallel streaming fluid boundary        during the sampling of the streams).    -   Synchronous and in phase laser motion scanning and parallel        streaming fluidic motion scanning such that the optical beam        moves in the same direction as the motion of the parallel        streaming fluid boundary (e.g. the analyzer takes one or more        consecutive samples of only the reference fluid, sample fluid or        fluid interface region over one or more cycles of a motion        waveform as may happen when the interrogation region motion is        the same as the fluid motion and thus the interrogation region        is stationary relative to the fluid interface boundary region).        This may be advantageous, for example, to sample the fluids or        interface region at different locations in the fluidic channel        as part sample measurement or calibration of the analyzer, all        without performing a ratioing or differencing of sample and        reference streams.    -   The fluid interface boundary region may be moved back and forth        across the fluidic channel and the laser beam may be motioned        controlled such that it continuously samples the sample,        interface (optionally) and reference fluidic regions at a motion        waveform frequency higher than the motion waveform frequency of        the parallel streaming fluidic motion translation. In this        manner, the differential measurement samples are taken at        multiple locations in the channel during laser beam motion        scanning. The frequency of the motion modulation may be an        integer multiple of the fluidic streaming modulation such that        an integer number of laser motion scanning reference and sample        measurements are taken for each cycle of the parallel streaming        boundary translation waveform.    -   As the fluid interface boundary in serial streaming travels        through the fluidic channel, the laser beam may be motioned        controlled such that it continuously samples the sample,        optionally interface, and reference fluidic regions (e.g. the        laser beam is translated during the measurement such that the        average location of the interrogation region during laser motion        scanning travels down the channel at the same rate as the        fluidic boundary. In this manner, measurement samples are taken        at multiple locations in the channel as the fluid travels down        the channel. The traveling laser beam motion may be performed in        1 or 2 dimensions (i.e. down and across the channel)    -   The fluid boundary is in motion with respect to the channel, and        the motion modulation signal includes a component that results        in a translation of the interrogation region correlated to the        motion of the fluidic boundary region.

Those versed in the art will recognized that the various embodiments ofthe fluid control and sensing techniques of this invention may alsoapply to non-optical techniques of measurement where it may beadvantageous to compare a sample and reference. Examples may includeconductivity measurements using electrodes or inductive loops,calorimetry, and pH. In such an example, the interrogation may not beoptical but instead is determined by the detection mechanism'sinteraction with the fluids, such as the electrical or magnetic pathwhen measuring conductivity.

While various embodiments have been particularly shown and described, itwill be understood by those skilled in the art that various changes inform and details may be made therein without departing from the scope ofthe invention as defined by the appended claims.

What is claimed is:
 1. A fluid analyzer, comprising: an optical sourceand an optical detector defining a beam path of an optical beam; a fluidflow cell disposed on the beam path defining an interrogation region ina fluid channel in the fluid flow cell in which the optical beaminteracts with fluids; one or more flow-control devices configured toconduct a particle in a fluid through the fluid channel; a motion systemconfigured to move the interrogation region relative to the fluidchannel in response to a motion signal; and a controller configured andoperative (1) to generate the motion signal having a time-varyingcharacteristic, (2) to sample an output signal from the optical detectorat a first interval of the motion signal during which the interrogationregion substantially contains the particle and at a second interval ofthe motion signal during which the interrogation region contains aregion substantially not containing the particle, thereby generatingcorresponding output signal samples, and (3) to determine from theoutput signal samples a measurement value indicative of an opticallymeasured characteristic of the particle.
 2. The fluid analyzer of claim1, including a feedback system having a measurement system for detectingthe location and motion of the particle within the fluidic channel, andwherein the detecting is used to calculate a new operating parameter ofthe controller.
 3. The fluid analyzer of claim 2, wherein duringoperation the fluid is in motion and the interrogation region is movedin the direction of fluid motion, and wherein the controller isconfigured and operative to (1) generate a second motion signal having atime-varying characteristic, (2) to sample a second output signal fromthe optical detector at a first interval of the second motion signalduring which the interrogation region substantially contains theparticle and at a second interval of the second motion signal duringwhich the interrogation region contains a region substantially notcontaining the particle, thereby generating corresponding second outputsignal samples, and (3) to determine from the second output signalsamples a measurement value indicative of an optically measuredcharacteristic of the particle.
 4. The fluid analyzer of claim 1,wherein a future motion signal is generated in response to themeasurement value.
 5. The fluid analyzer of claim 1, wherein the fluidcontains multiple particles in the interrogation region and themeasurement value is indicative of an optically measured characteristicof the multiple particles.
 6. The fluid analyzer of claim 1, wherein theone or more flow-control devices is configured to conduct a second fluidin the fluid channel, and the motion signal is operative to position theinterrogation region in the first fluid during the first interval and inthe second fluid during the second interval.
 7. The fluid analyzer ofclaim 6, wherein the first and second fluids are flowing in the fluidchannel during the first and second intervals respectively.
 8. The fluidanalyzer of claim 7, wherein the first fluid flow is terminated betweenthe first and second intervals respectively.
 9. The fluid analyzer ofclaim 6, wherein a flow of the first or second fluid in the fluidchannel is terminated prior to the first or second interval.
 10. Thefluid analyzer of claim 6, wherein the motion control device conducts athird fluid into the fluid channel, the third fluid spatially betweenthe first and second fluid.
 11. The fluid analyzer of claim 1, whereinthe volume of first fluid consumed in the measurement is materially lessthan the volume of the second fluid.
 12. The fluid analyzer of claim 1,wherein the interrogation region in the second interval contains aregion containing a second particle.
 13. The fluid analyzer of claim 12,wherein the first interval interrogation region contains more or fewerparticles than the second interval interrogation region.
 14. The fluidanalyzer of claim 13, wherein the measurement value is indicative of thedifference in optical signal sampled at the detector in the first andsecond interval.
 15. The fluid analyzer of claim 12, wherein the firstor second interval interrogation region contains a known distribution ofparticles.
 16. The fluid analyzer of claim 1, wherein the optical beamsignal incident on the detector has been spatially filtered for thepurpose of detecting scattered optical signal.
 17. The fluid analyzer ofclaim 1, wherein the optical beam signal incident on the detector hasbeen spatially filtered for the purpose of removing scattered opticalsignal.
 18. The fluid analyzer of claim 1, wherein measurement valuesare determined at multiple wavelengths of optical light to estimateparticle size.
 19. The fluid analyzer of claim 1, wherein the opticallymeasured characteristic of the particle is an optically measuredcharacteristic of an interaction of the particle and the surroundingfluid.
 20. The fluid analyzer of claim 1, wherein the optically measuredcharacteristic of the particle is an optically measured characteristicof an interaction of the particle and the optical beam.
 21. The fluidanalyzer of claim 1, wherein the optically measured characteristic ofthe particle is an optically measured characteristic of an interactionof the particle and the microfluidic cell.
 22. The fluid analyzer ofclaim 1, wherein the fluid channel supports multiple simultaneousoptical beams and interrogation regions, and the multiple interrogationregions are sampled by the controller.
 23. The fluid analyzer of claim1, wherein the controller is operative to adjust the position, size orshape of the interrogation region.
 24. The fluid analyzer of claim 23,wherein a position, size or shape of the interrogation region isdetermined by the controller in a feedback loop that uses themeasurement value.
 25. The fluid analyzer of claim 1, wherein the firstfluid contains a particle with a known value of an optically measuredcharacteristic, and (1) a measurement value of the particlecharacteristic and (2) the known value for of an optically measuredcharacteristic are used in determining an optically measuredcharacteristic of the particle.
 26. The fluidic analyzer of claim 1,wherein the first fluid contains a dissolved analyte, and measurementvalue at multiple wavelengths of the optical beam are used to determinea measurement value of the analyte.
 27. A fluidic analyzer of claim 1,wherein the measured values at multiple wavelengths of the optical beamare used to estimate particle chemical composition and size.
 28. Thefluid analyzer of claim 1, wherein the motion signal provides for rapidspatial scanning of the interrogation region over dimensions on theorder of the particle size and slower spatial scanning over dimensionssubstantially greater than the particle size.
 29. The fluid analyzer ofclaim 28, wherein the rapid spatial scanning is a 1-dimensional scan andthe slower spatial scanning is a 2-dimensional scan.
 30. The fluidanalyzer of claim 28, wherein the slower spatial scanning is in a regionwith a known distribution of included particles or droplets.
 31. Thefluid analyzer of claim 1, further comprising a channel imaging devicewith an output signal used by the controller for determining the motionsignal and the first and second intervals.
 32. The fluid analyzer ofclaim 1, further comprising a transducer with a transducer output signalused by the controller and indicative of the presence of the particleand its position in the channel.
 33. The fluid analyzer of claim 1,wherein the particle is adhering to a surface of the fluid channelduring the first interval.
 34. The fluid analyzer of claim 1, whereinboth transmission and emission detection are performed, wheretransmission is used to detect immiscible analytes and infrared emissionis used to detect or image non-immiscible analytes.